Rifamycin antibiotics - New compounds and synthetic methods. Part 3: Study of the reaction of 3-formylrifamycin SV with primary amines and ketones

Article abstract of DOI:10.1016/j.tet.2012.04.071

In the third stage of our study concerning the search for new antibacterial rifamycin antibiotics, the reactions of 3-formylrifamycin SV (1) with a range of primary alkylamines and ketones of general structure R₁-CH ₂-CO-R₂ (R₁H or alkyl and R₂alkyl or aryl) has been investigated. A new synthetic method for the preparation of a new group of rifamycin derivatives with an α,β-unsaturated imine substituent at C-3 has been developed. These compounds showed a tendency to reversibly isomerise in organic solvents and, in the presence of water, to rapidly hydrolyse. The structures of four isolated microcrystalline compounds 2, 3, 4, 5 and a reaction's mechanism have been proposed on the basis of mass spectrometry results as well as (1D) and (2D) ¹H and ¹³C NMR analysis. The new synthetic route reported herein is a promising pathway to new reactive rifamycins displaying broader capabilities than the plain 3-formylrifamycin SV.

Full text of DOI:10.1016/j.tet.2012.04.071

Tetrahedron 68 (2012) 5925e5934
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Rifamycin antibioticsdnew compounds and synthetic methods. Part 3: Study
of the reaction of 3-formylrifamycin SV with primary amines and ketones
, ,
,
,
b
Krzysztof Bujnowski a y, Ludwik Synoradzki a y, Thomas Zevaco b , Eckhard Dinjus
*
*
a Warsaw University of Technology, Faculty of Chemistry, Laboratory of Technological Processes, ul. Noakowskiego 3, 00-664 Warsaw, Poland
b Institute of Catalysis Research and Technology, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In the third stage of our study concerning the search for new antibacterial rifamycin antibiotics, the
reactions of 3-formylrifamycin SV (1) with a range of primary alkylamines and ketones of general
structure R1eCH2eCOeR2 (R1]H or alkyl and R2]alkyl or aryl) has been investigated. A new synthetic
Received 10 February 2012
Received in revised form 17 April 2012
Accepted 19 April 2012
method for the preparation of a new group of rifamycin derivatives with an
substituent at C-3 has been developed.
a,b-unsaturated imine
Available online 4 May 2012
These compounds showed a tendency to reversibly isomerise in organic solvents and, in the presence of
water, to rapidly hydrolyse. The structures of four isolated microcrystalline compounds 2, 3, 4, 5 and
a reaction’s mechanism have been proposed on the basis of mass spectrometry results as well as (1D) and
(2D) 1H and 13C NMR analysis. The new synthetic route reported herein is a promising pathway to new
reactive rifamycins displaying broader capabilities than the plain 3-formylrifamycin SV.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Antibiotics
Ansa compounds
Aldol condensation of imines
NMR spectroscopy
a,b-Unsaturated imines/isomerisation
1. Introduction
In the first synthesis’ step, 3-formylrifamycin SV (1) was, however,
subjected, not to the aldol reaction but to the Wittig reaction with
Rifamycins belong to the class of ansamycins,1 playing an im-
portant role as antibiotics in therapeutics against tuberculosis,
leprosy and various other mycobacterial infections.2,3 The most
efficient and well established rifamycins are rifampicin, rifabutin,
rifapentine and rifaximin,4 however the increasing microbial re-
sistance found with these commercially available rifamycins calls
for new therapeutic strategies and/or new rifamycin sorts.5,6 In the
first part of our work dealing with new antibacterial rifamycins, the
products of the reaction of 3-formylrifamycin SV (1) with ammonia
or primary alkylamines were investigated.7,8 In the second part, we
characterized the derivatives obtained from the reaction of rifa-
mycin 1 or 3-formyl-25-O-desacetyl rifamycin SV with ammonia
and acetone.9,10
phosphorus ylides, e.g., acetylemethylenetriphenyl phosphine,
obtaining various 3-(3-oxo-alkylidenyl)erifamycins SV. In a sub-
sequent synthesis step these derivatives were condensed with ap-
propriate
hydroxylamine-,
phenylhydrazine-,
N-amino-N0-
alkylpiperazine- or hydrazine derivatives, obtaining new com-
pounds displaying an enlarged substituent at carbon C3 of the
chromophore, with Schiff base, hydrazone or oxime characteristics.
All these new groups of derivatives were tested in view of antibac-
terial and antiviral activity. Some substances, especially the oxime
derivatives, showed good activity against gram-positive bacteria,
comparable to that of already reported classes of rifamycins. The
three most effective derivatives showed good inhibiting action to-
wards several RDDP (RNA-dependent DNA polymerases) chosen for
The third part of this work concerns the study of the reaction of
3-formylrifamycin SV (1) with selected primary alkylamines and
ketones of general structure R1eCH2eCOeR2, where R1]H or alkyl,
virus tests (inhibition level of 50e80% at concentrations 20 mg/ml).
In the published patent,13 Cricchio claimed, besides the method
of obtaining 3-(3-oxo-alkylidenyl)-rifamycins SV via the use of
corresponding ylides, also the synthesis of these rifamycin SV de-
rivatives via an aldol-like synthesis reacting 1 with ketones dis-
playing a R1eCH2eCOeR2 structure, where R1]H or alkyl and R2]
H, alkyl, aryl. However this second method was only illustrated
with one example, where 1 condensates with acetaldehyde, the
reaction running in tetrahydrofurane in the presence of pyridine
and acetic acid at ca. 5 ꢀC. Cricchio mentioned various bases, in-
cluding alkali metals hydroxides, alkoxides and amines as well as
ammonium acetate or alkali metals acetates as potential catalysts
for this kind of reactions.
R2]alkyl, aryl. The synthesis of related a,b-unsaturated ketones via
cross aldol synthesis (ClaiseneSchmidt condensation) of one of the
3-alkenylrifamycin groups was the initial goal of these studies.11
Several groups of 3-alkenylrifamycin SV derivatives were pro-
duced in the 70’s of the last century by R. Cricchio and co-workers.12
* Corresponding authors. E-mail addresses: bujba@ch.pw.edu.pl (K. Bujnowski),
thomas.zevaco@kit.edu (T. Zevaco).
y
Tel.: þ48 22 6210138; fax: þ48 22 6255317.
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.04.071

5926
K. Bujnowski et al. / Tetrahedron 68 (2012) 5925e5934
2. Results and discussion
The presence of ‘acidic’ protons in a position of the carbonyl
group is paramount to observe a significant reaction of aldehyde 1
with primary amines and ketones, as confirmed by the experiments
carried out. However, in a ‘standard’ cross aldol reaction, an amine
usually plays the part of the catalyst: abstracting a proton from the
2.1. Generalities on the reaction
In order to perform a screening of the cross aldol reaction with
3-formylrifamycin SV (1, Fig. 1), we selected aliphatic and alipha-
ticearomatic ketones (Table 1) bearing in their structure at least
one methylene or methyl group in
According to the literature, the presence of this methylene group is
a prerequisite for a dehydration of the intermediately formed
methylene in C-a
of the ketone to form a reactive carboanion.9,15 It
was found in our case that, amines, besides activating the acidic
methylene group, are also directly engaged in the reaction, forming
a position of the carbonyl group.
reactive
a,b-unsaturated imines. Therefore, various primary ali-
b
-
phatic amines, with linear, branched and cyclic alkyl groups, were
used to assess the influence of the amine’s substituents on the re-
action progress (vide infra, section 2.3).
hydroxyketones to eventually yield the desired
a,b-unsaturated
ketone derivatives (vide infra, section 2.4).14
A wide range of polar solvents were also tested, revealing that
lower alcohols, and especially methanol, are the most suited sol-
vents to carry out the synthesis. A rapid reaction together with
a good selectivity and easy crystallization of the main product were
also observed when using ethylene glycol as solvent, nevertheless
the complete removal of this solvent during the drying step
revealed to be quite tedious. In some cases it was also possible to
run the reaction and crystallize the product in chloroform. In all
cases, the reaction has to be performed in a narrow temperature
window: at temperatures lower than 35 ꢀC the reactions proceeded
very slowly or did not proceed at all, whereas above 45 ꢀC the
36
H3C
O
33
32
31
35
CH3 CH3 CH3
25
O
21
24
22
20
19
26
27
37
OH
O
28
OH
CH3
18
17
16
H3C
34
O
OH OH
CH3
15
NH
29
30
14
1
8
H3C
9
7
6
2
3
10
H
5
4
O12
11
OH
O
O
O
hydrolysis of imines E to the corresponding
tones F considerably increased.
a,b-unsaturated ke-
CH3
13
The addition of an acid had a positive effect on the rate of the
reaction, which is in agreement with the related literature.16 Among
the common carboxylic and sulfonic acids tested as catalysts (acetic
C38H47NO13
M 726
1
Fig. 1. Structure of 3-formylrifamycin SV.
acid, trichloroacetic acid,
L-tartaric acid, citric acid, methanesulfonic
acid, p-toluenesulfonic acid), the best results were obtained with
Table 1
List of the investigated ketones with the final product types (E, B, F as in Scheme 1 or complex mixture: X)

K. Bujnowski et al. / Tetrahedron 68 (2012) 5925e5934
5927
acetic acid. The action of the acid is discussed more in detail in the
paragraph dealing with the reaction mechanism (section 4).
Reaction progress and purity of the isolated rifamycin de-
rivatives were controlled by thin layer chromatography (TLC), an
‘old-fashioned’ analytic method, which works surprisingly well in
the tracking and quantification of antibiotics.17,18
retention coefficients, displaying usually the arrangement
RfB>RfE2>RfE1>Rf1>RfF.
The reaction was regarded as completed when substrate 1 was
consumed and the concentration of intermediate imine B was low.
Afterwards the reaction mixture was cooled to 0e5 ꢀC and usually
left at this temperature for about 12 h. In many cases a ‘navy-blue’
main product was obtained identified as type E2 imine (TLC), which
was filtered off, washed with cold methanol, and then dried under
vacuum at 40 ꢀC. When 4-methyl-2-pentanone (isobutyl-methyl
ketone) was used in the reaction, type E2 imines did not crystallize
from methanol, even at low temperature, and blue imines of E1 type
were the dominating products in the reaction mixture. The final
reaction mixture was diluted with water and extracted with chlo-
roform. A number of blue microcrystalline imines of the E1 type
were obtained from the chloroform concentrates. Unfortunately, in
neither cases, E1 or E2, was it possible to isolate single crystals good
enough for a supporting X-ray diffraction structural determination.
On the whole, it was found that:
In solutions containing E2 imines (solvent: MeOH, CHCl3, THF
and DMSO), an isomerisation of these compounds proceeds giving
E1, as recorded via TLC where besides a dark blue spot of E2, a blue
spot of E1 increasingly appears. The rate of this isomerisation is re-
lated to the structure of imine E2, allowing in some cases to study
comprehensively the structure also via time-consuming NMR-
techniques (e.g., 2D correlation spectroscopies). The 1H and 13C NMR
spectra in DMSO-d6 of the slowly isomerising imine 4 of type E2, 3-
(30-isopropylimino) nonenylrifamycin SV, (Fig. 3) were recorded at
ambient temperature at a time not exceeding 4 h, shows one type of
imine in solution. Overnight the same sample clearly exhibits two
sets of signals characteristic of a mixture of isomers. Conversely, in
analogous solutions of imine E1, the E2 isomer slowly appears.
2.2. Evolution of the reaction and isolation of the main
products
According to the standard procedure, the solvent (mainly
methanol) was poured into the reaction flask, followed by the ad-
dition of acetic acid, amine, 3-formylrifamycin SV (1) and the re-
spective ketones, the mixture being stirred around 40 ꢀC for one to
4 h. In a typical reaction’s course, the control chromatograms
(TLCdsee experimental part) of the reaction mixture showed over
the usual reaction time a gradual decay of the initial red spot
characteristic of the 3-formylrifamycin SV and the appearance of:
(i) first a violet spot, probably related to an intermediate imine of
structure B, (Scheme 1) and (ii) an intense blue spot of product E1,
which slowly dominated in the system.
36
33
32
31
H3C
O
CH3 CH3 CH3
35
O
25
23
21
24
19
22
20
26
27
37
H3C
O
OH OH
CH3
18
17
34
28
OH OH
16CH3
O
15
29
30
14
H3C
1
8
NH
9
7
H
2
3
6'
CH3
2'
10
5
3'
4'
5'
6
1'
4
O
11
12
OH
H
O
CH3
6'
O
O
CH3
13
C44H57NO13
M = 807
6
Fig. 2. Structure of the
a,
b-unsaturated ketone 6 of type F.
The presence of water traces in the solutions, besides the de-
Scheme 1. Mechanism proposal for the reaction of 3-formylrifamycin SV (1) with
primary amines and ketones.
scribed E14E2 isomerisation, leads to a slow hydrolysis of E1/E2 to
yield F (a,b-unsaturated ketone). Another product B (most likely also
an imine) appears as a result of a gradual conversion of E in B (TLC).
Ketones of type F (e.g., compound 6: 3-(50-Methyl-30-oxo)hex-
enylrifamycin SV; Fig. 2) do not react in the presence of primary
amines to yield imines of type E again, indicating that the hydro-
lysis of E is not reversible.
This fraction was identified as a rifamycin SV derivative with an
a,b-unsaturated imine substituent at C-3. Simultaneously, the for-
mation of the second product, E2, affording a navy blue spot on the
chromatogram (usually smaller than that of E1) was observed, E2
being an isomer of E1 (Scheme 1).
Substance E2dslightly soluble in methanol, started to crystal-
lize, sometimes already in the reaction mixture. In a further re-
action step imines E1 and E2 underwent a gradual hydrolysis to
2.3. Effect of amine concentration and structure
form the
the chromatogram.
Fortunately the various substances spotted using the TLC
method could be easily differentiated thanks to the broad range of
a
,
b
-unsaturated ketone F, giving a burgundy-red spot on
In the study at hand, primary alkylamines with linear chains (n-
propylamine, n-butylamine and n-hexylamine), branched chains
(isopropylamine and isobutylamine) and cyclic chain (cyclohexyl-
amine) as well as hydroxylalkylamined(ethanolamine) were used.

5928
K. Bujnowski et al. / Tetrahedron 68 (2012) 5925e5934
36
36
33
32
31
H3C
O
H3C
O
33
32
31
CH3 CH3 CH3
35
35
CH3 CH3 CH3
25
25
O
23
21
19
20
O
21
24
22
24
22
20
19
26
26
27
37 O
OH OH
18
37 O
H3C
OH
OH
CH3
18
17
17
H3C
27 CH3
34
34
28
OH OH
16CH3
OH OH
16CH3
28
O
O
15
15
NH
29
30
29
30
14
H3C
14
8
1
1
8
NH
9
H3C
7
6
9
7
6
2
2
6'a
6'b
6'a
6'b
3 H3C
CH3
10
3 H3C
CH3
10
5
5
5'
4'
5'
4'
4
1'
4
1'
2'
O12
O12
11
11
2'
OH
O
3'
OH
O
3'
O
O
2"a
CH3
CH3
CH3
13
N
N
13
1"
1"
2"
CH3
H3C
2"b
3"
2 (E1)
C47H64N2O12
M = 848
3 (E1)
C47H64N2O12
M = 848
36
36
33
32
31
H3C
O
33
32
31
H3C
O
CH3 CH3 CH3
35
CH3 CH3 CH3
35
25
O
23
21
25
O
23
21
19
24
22
20
19
15
24
22
20
26
27
26
27
37 O
OH OH
CH3
18
37 O
H3C
OH OH
CH3
18
17
16
17
16
H3C
34
34
28
28
O
O
OH OH
CH3
15
OH OH
CH3
30
29
30
14
H3C
29
14
H3C
1
8
1
8
NH
7
6
9
NH
7
9
2
3
2
3
10
5
10
5
6'
5'
7'
6
6'
5'
7'
4
1'
2'
O
4
1'
2'
O
11
12
11
12
OH
4'
CH3
O
3'
OH
4'
8'
O
3'
8'
CH3
O
O
9'
2"a
CH3
6"
CH3
13
13
N
H3C
N
5"
4"
1"
2"
1"
CH3
2"b
3"
5 (E2)
4 (E2)
C49H70N2O12
M = 878
C51H70N2O12
M = 902
Fig. 3. Structures of four representative a,b-unsaturated imines of type E1 and E2.
3. The reaction stopped at the step of imine B.
All the amines studied formed products with a general iminic
structure E, albeit the imimes formed using ethanolamine showed
the greatest tendency towards fast hydrolysis. The best yield and
selectivity towards imines of type E was achieved when using
5 mmol of amine per 1 mmol of 1. An amine deficit caused the
suppression of the B to E transformation (Scheme 1).
To find a relationship between structure of the ketones and
reaction progress, comparative attempts of reacting 1 with n-
butylamine and a broad range of selected ketones were performed
(see Table 1 for overview).
Symmetric dialkyl ketones as well as cyclic ketones like cyclo-
pentanone, cyclohexanone and 4-methylpiperidone were also
tested in the presence of acetic acid in methanol. To extend this
series two alkyl-aryl ketones, p-methylacetophenone and 2,4-
dimethyl-acetophenone were also used in this synthesis. These
studies were also supplemented in some cases by reacting 1 with
definite ketones and other primary alkylamines.
On the basis of chromatographic monitoring (TLC) of the re-
action and some NMR knowledge of the system (vide infra, section
3), the following observations were made:
2.4. Effect of ketone concentration and structure
In a majority of cases, the optimal yields for the imines E were
achieved when using about 2.5 mmol of ketones for 1 mmol of 1.
Although an increase of the ketone amount usually accelerates the
formation of imines, this procedure was only used in cases where
the synthesis proceeded too slowly. The reason for this is that
a larger amount of unreacted ketone in the reaction mixture af-
fected negatively the crystallization of imine E2 from this mixture.
The nature of the alkyl substituent in the methylealkyl ketones
shows a considerable effect on the synthesis of rifamycins dis-
playing an a,b-unsaturated imine structure E (Table 1 for an over-
It was found that the synthesis’ efficiency of a,b-unsaturated im-
ines (E1 or E2) was related to the nature of the ketone. Three main
ways of reaction can be distinguished for the investigated system
(Table 1):
view of the reaction’s outcome):
1. When a methylene group is present in
a position of the car-
1. The formation of imines E1 or E2 proceeded quickly, the
products crystallizing rapidly from the reaction mixture in the
case of E2, or, considering product E1, from the concentrated
chloroform extract (see 2.2).
bonyl carbon, the reaction proceeded very rapidly and the de-
sired productd -unsaturated imine E was formed. This
a,b
compound underwent in some cases (with e.g., 2-butanone, 2-
pentanone 2-hexanone or benzylemethyl ketone) a rapid hy-
drolysis to a,b-unsaturated ketone F and the further isolation of
2. The reaction delivered no crystalline materials: neither product
E1 nor E2 was obtained. This is most likely due to a partial
the crystalline imines failed. However, some imines E2 (based
on 2-heptanone and 2-octanone) as well as of imines E1
hydrolysis to form
crystallisation of E2 or E1.
a,b-unsaturated ketone F that hinders the

K. Bujnowski et al. / Tetrahedron 68 (2012) 5925e5934
5929
(involving e.g., 4-methyl-2-pentanone), could be isolated. The
C]C double bond in the new substituent at carbon C-3 of the
isolated compounds was always preferentially formed
involving the methyl group carbon of these ketones (NMR).
and similarly imine 3, product of the reaction of 1 with iso-
propylemethyl ketone and propylamine, provided very clear NMR
spectra due to a high purity and exceptional stability in DMSO-d6.
Together with mass spectrometry and IR studies, the proposed
structure of this compound could be determined (Fig. 3).
2. When a methine group is present at the
a position, as e.g., in 3-
methyl-2-butanone, the formation of imine E proceeds much
slower than that in the above described system. This product
undergoes relatively rapidly hydrolysis to yield F, and hence
crystalline imines E, derivatives of such ketones, have not been
isolated.
On the other hand, NMR spectra in DMSO-d6 of various type E2
imines, despite a high purity (TLC) of the materials, were quite
complex due to an overlapping of the signal of both isomers, the
second isomer being rapidly formed in solution.
This confirmed the isomerisation of E2 in E1 observed by means
of TLC. Working under argon and with ‘bone-dry’ deuteriated sol-
vent, it was possible to gather enough exploitable NMR data of the
type E2 imines, 4 and 5, to establish their structures. To supplement
the analytical studies performed on type E1 and E2 imines, a similar
3. When a quaternary carbon atom is present in the alkyl group in
a
position as, e.g., in methyl-tert-butyl ketone, only the for-
mation of imine B can be observed as well as the formation of
the -unsaturated ketone, F.
a,b
NMR analysis was run on a a,b-unsaturated ketone of type F, 6,
From the above observations it results that increasing the steric
hindrance of the group bound to the ketone hinders the formation
which confirmed the proposed structure.
In general, the assignment of NMR signals in 1H and 13C spectra of
rifamycins 2, 3, 4, 5 and 6 (hydrolysis product of 2) was made corre-
lating the information of 1D and 2D-NMR spectra: 1H,1H COSY, 1H,13C
HSQC and 1H,13C HMBC. In the case of 2, DEPT spectra were addi-
tionallyrecordedtogetherwith1Hspectrabeforeandafteradditionof
D2O to identify the mobile protons of the naphthol chromophore. The
recorded chemical shifts were compared with literature data con-
cerningNMRstudiesofrifamycins20e25 andourownNMRdatabaseof
rifamycin derivatives.7,8 To avoid needless redundancy, we will only
describe in detail the structure characterization of one E1 derivative
(2) and of a related hydrolysis product, 6. The complete character-
ization of derivatives 3, 4 and 5 can be found in the Supplementary
data together with the related NMR-correlation tables.
of the imine E, probably due to an enhanced affinity of the
aminoimine D towards hydrolysis.
b-
Interestingly only the alkyl-methyl ketones used in the study
formed a product displaying an imine E structure, in other cases the
synthesis seems to be stopped at stage B. Acetone as a particular
case reacts rapidly to yield the related
From the different methyl-aryl ketones investigated only 2,4-
dimethylacetophenone yield the desired -unsaturated imine E
a,b-unsaturated ketone F.
a,b
in crystalline form; the others ketones delivering only complex
mixtures after rapid reaction with 1 and n-butylamine.
Cyclopentanone and cyclohexanone yield, in the reaction of 1
with monoalkylamines, a mixture of compounds which could not
be separated. Only 4-methylpiperidone reacted with 1 and butyl-
amine to yield the corresponding
was isolated as microcrystalline solid.
On the whole, these studies resulted in the synthesis and iso-
-unsaturated
a,b-unsaturated ketone F, which
3.2. NMR study of rimamycin 2: properties of the substituent
at carbon C-3 (Fig. 3, Table 2)
The signal at 117.1 ppm in the 13C NMR spectrum was assigned to
C-10. In the 1H NMR spectrum the signal of the 10-H proton occurs as
a doublet at 7.94 ppm. The signal at 148.7 ppm (overlapping with
the C-1 signal) was assigned to C-20. In the 1H NMR spectrum, the
signal of 20-H at 7.81 ppm appears as a doublet. In the 1H,1H COSY
spectrum a clear correlation signal 10-H/20-H J1’,2’¼15.6 Hz was
found. The high coupling constant indicates a trans geometry of
protons 10-H and 20-H.26a On the basis of the correlations found in
2D-NMR spectra, the low-field signal at 177.9 ppm was assigned to
the imine carbon atom C-30, the signal being located in the region
characteristic of the iminic C]N bond (w150e180 ppm).26b The
methylene group C-40 was associated to the signal at 40.7 ppm
(phased-down in DEPT 135 spectrum). The magnetically non-
equivalent methylene group 40-Ha and 40-Hb protons in the 1H
NMR spectrum afford multiplets at 2.68 and 2.24 ppm, respectively.
In the 13C NMR spectrum the signal at 28.9 ppm was assigned to C-
50, and related to the signal at 2.00 ppm (50-H), overlapping the
signals of 30-3H and 36-3H. The signals at 22.2 and 20.8 ppm in the
13C NMR spectrum were assigned to the non-equivalent methylene
group carbon atoms C-60a and C-60b, respectively, and the signals at
0.83 and 0.77 ppm in the 1H NMR spectrum were assigned to the
60a-3H and 60b-3H protons of these methylene groups. This non-
equivalency of carbon C-6a0 and C-60b atoms correlated by the pro-
ton data, indicates clearly an inhibition of the rotation around bond
C-40eC-50, caused by the steric hindrance of the neighbouring
isopropyl group at the imine nitrogen (Table 2).
lation as microcrystalline materials of a number of a,b
imines of structure E (N-substituted 3-(30-imino)alkylidenylrifa-
mycins SV, yields ranging from 20 to 70%). In many cases, the final
products isolated from methanol displayed traces of isomers E2,
besides the major product isomer E1.
In order to convert the isolated imines E1 and E2 into the related
a,b-unsaturated ketones F, they were heated in slightly alkaline
aqueous-methanol solutions (see experimental part) until the
chromatographic monitoring of the mixture showed a conversion
of E to F. Products of structure F were most often obtained in
a
crystalline form from chloroform solutions. This synthetic
method, yielding ketones from imines of structure E is much easier
than that described formerly in the literature.20
3. Analytical studiesddetermination of the structure of
rifamycins 2, 3, 4, 5 and of the hydrolysis product 6
A comprehensive analytical study is somewhat hindered by the
labilityofsomeof theintermediates. TheisomericiminesoftypeE1or
E2, isolated from the reaction mixtures, generally have a microcrys-
talline structure and can contain traces of impurities, mainly the
second isomer, or rarely, the hydrolysis products, the corresponding
a,b-unsaturated ketones F. The general low solubility in organic sol-
vents (possibly due to the formation of stable rifamycin-solvent ad-
ducts during thesynthesis19), aswell asthe highreactivityoftheC]N
double bond prone to isomerisation and hydrolysis, might be an ex-
planation for the evident difficulty to get single crystals suitable for X-
ray diffraction studies.
The signal at 48.6 ppm was assigned to the ternary C-100 carbon
atom bound to the imine nitrogen atom of the substituent at C-3.
The signals of methylene groups C-20a0 and C-2b00 carbons overlap at
21.1 ppm, whereas the protons’ signals of these groups, 20a0-3H i
20b0-3H, are separated, appearing as doublets at 1.31 and 1.26 ppm.
This suggests a constrained geometry around the NeC-100 bond via
the presence of a bulky alkyl fragment at C-30.
3.1. NMR studies
Among several crystalline E1 imines, the imine 2, product of the
reaction of 1 with isopropylemethyl ketone and isopropylamine,

5930
K. Bujnowski et al. / Tetrahedron 68 (2012) 5925e5934
Table 2
13C and 1H NMR data of 2 in (d6)DMSO
Atom C
C-1
D
Atom H
(amide)eH
d
Multipl.
s
JH,H
1H,1H COSY correlations
1H,13C HMBC correlations
C-1, C-2, C-15
N
9.07
148.7a
1-OH
15.75
14.14
s
C-1, C-2
C-2
C-3
C-4
116.6
119.0
151.7
4-OH
s
C-3, C-4
C-5
C-6
C-7
C-8
99.2
171.6
103.3
183.7
8-OH
11.59
br
10’-H lr.
C-9
117.3a
117.3a
185.5
108.7
21.9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
3 13-H
3 14-H
1.63
1.90
s
s
C-11, C-12
C-6, C-8
7.3
168.1
131.5
131.9
125.4
139.5
37.8
17-H
18-H
19-H
20-H
21-H
21-OH
22-H
23-H
23-OH
24-H
6.34
6.78
6.04
2.24
3.63
5.08
1.21
2.75
3.88
1.54
5.05
0.96
3.20
4.86
6.24
1.92
0.67
0.35
0.85
ꢁ0.34
d
(17,18)¼11.2
(18,19)¼16.0
(19,20)¼7.0
18-H, 3 30-H lr.
17-H, 19-H
18-H
21-H, 3 31-H
20-H, 21-OH
21-H
23-H, 3 32-H
22-H, 23-OH
23-H
3 33-H
26-H
25-H, 3 34-H
28-H
27-H, 29-H
28-H
dd
dd
ma
m
da
ma
ma
d
71.8
(21-OH,21)¼3.6
C-21, C-22
C-24
C-22
C-23
38.1
75.6
(23-OH,23)¼8.8
(25,26)¼10.8
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
32.4
73.1
40.3
76.2
117.6
143.0
20.5
17.6
8.8
m
dd
ma
d
25-H
26-H
27-H
28-H
C-23, C-24, C-26, C-34 C-35
(27,28)¼8.8?
(28,29)¼12.8
dda
d
29-H
C-12, C-27, C-28
C-16
C-19, C-20, C-21
C-22, C-23, C-25
C-21, C-23, C-24
C-26, C-27
3 30-H
3 31-H
3 32-H
3 33-H
3 34-H
sa
17-H lr.
20-H
22-H
24-H
26-H
d
(31,20)¼6.4
(32,22)¼6.4
(33,24)¼6.8
(34,26)¼6.4
d
10.8
8.9
da
d
169.2
20.0
55.5
117.1
148.7a
177.9
40.7
3 36-H
3 37-H
10-H
1.92
2.84
7.94
7.81
sa
s
d
d
C-35
C-27
C-2, C-30
C-10, C-30
(10,20)¼15.6
20-H
10-H
C-20
20-H
C-30
C-40
40-Ha
2.68
2.24
2.00
0.83
0.77
4.24
1.31
1.26
dda
40-Hb, 50-H
40-Ha, 50-H
a
40-Hb
a
C-50
28.9
22.2
20.8
48.6
21.1a
21.1a
50-H
40-Ha, 40-Hb, 3 60a-H, 3 6b0 -H
50-H
C-60b
C-6’a
C-6’b
C-100
C-20a0
C-20b0
3 60a-H
3 60b-H
100-H
da
d
m
da
da
(60a,50)¼6.4
(60b,50)¼6.4
(100,8-OH)z3.0
(20a0,100)¼6.4
(20b0,100)¼6.4
C-50, C-60b
C-50, C-60a
50-H
8-OH lr., 3 20a0-H, 3 20b0-H
100-H, 3 20b0-H
100-H, 3 20a0-H
3 20a0-H
3 20b0-H
C-100, C-20b0
C-100, C-20a0
Abbreviations: s: singlet, d: doublet, m: multiplet, br: broad, lr: long range correlation.
a
This signal overlapped with another signal.
1
3.3. NMR study of rimamycin 2: characterization of the ansa-
backbone
the H,1H COSY spectrum, the low-field position of 10-H signal
(4.24 ppm) and the small difference in chemical shifts of carbon C-8
(183.7 ppm) and C-11 (185.5 ppm) signals result probably from
a tautomeric equilibrium, analogous to that described in details in
our previous publication.9
In the low-field part of the 1H NMR spectrum (16.0e8.0 ppm)
the signals of four mobile protons (all disappear after H/D ex-
change) were found. The signal at 15.75 ppm (s) is assigned to the
1-OH proton. In the 1H,13C HMBC spectrum 1-OH/C-1, 1-OH/C-2
correlations are observed. The proton giving a signal at 14.14 ppm,
showing a long range coupling with C-3 (119.0) and C-4 (151.7),
belongs to the 4-OH hydroxyl group. The signal at 9.07 ppm in the
1H NMR spectrum, showing long range coupling with the C-1
(148.7 ppm) and C2 signals (168.1) was assigned to the amide group
proton N(amide)eH. The remaining broad signal (11.59 ppm) was
assigned to the 8-OH proton. The 8-OH/10-H correlation observed in
Considering the whole NMR data set, none of the analytically
studied compounds, either E1- or E2-type rifamycin, displayed an
enamine structure. This is quite obvious studying HMBC and HMQC
spectra of the compounds. On the one hand, no 1H-signals char-
acteristic of enamine protons eCH]C-30eNHeR could be detected
and, on the other hand, clear C/H correlations were observed in-
dicating the presence of two protons at C-40 carbon.
The substituents containing protons 10-H and 20-H, in both 2 and
3 (blue imines of type E1) as well as in 4 and 5 (dark blue imines of

K. Bujnowski et al. / Tetrahedron 68 (2012) 5925e5934
5931
type E2) have a trans arrangement related to the double bond
C-10]C-20.
imine B, observed chromatographically (TLC). The structure of
similar imines was determined in earlier studies on the conden-
sation of 1 and monoalkylamines.7
Simultaneously, the starting ketone present in the system forms
an imine C via acid-catalyzed reaction with the starting alkyl-
amine.16,27 The reaction of imines B and C in the following stage
It seems reasonable to think that having two possible configu-
rations at the imine bond C-30]N (syn (Z) or anti (E)) explains the
presence of the two isomeric forms E1 and E2. According to Kali-
nowski,26b the NMR signals of the C]N bond found in isomeric
imines display, for the anti configuration a low-field shift of the
leads to the formation of a
quickly rearranges to eliminate an alkylamine molecule, thus
forming the -unsaturated imine E. This imine occurs in the re-
b-amino-imine D. This transient specie
carbon in a
-position of the imine carbon, i.e., C-40 in 2e5, relative to
the signal recorded for the the syn (Z) arrangement.
a,b
Prolonging the recording time of the 13C NMR spectrum of 5
(max. 30 h), enabled the E2/E1 isomerisation to take place, giving
a second set of signals. Especially a clear decrease in the E2 signals
assigned to C-40 (27.0 ppm) and of C-60 (29.0 ppm) and the appear-
ance of two distinct new signals at 29.5 ppm and 32.9 ppm, assigned
tothe structure E1 couldbe noticed. Interestingly in drysolutions, no
hydrolysis from E to F occurred as confirmed by the lack of NMR
signal characteristic of a ketone ca. 200 ppm. Hence, it seems logical
that the configuration of the double bond in imines 5 and 4 as well as
in other E2 type imines is of the anti type and, conversely the double
bond in type E1 imines should display a syn configuration.
action mixture in two isomeric forms, E1 and E2, however, a re-
versible E14E2 isomerisation proceeds here. In many cases, the E2
isomer, weaklysoluble in methanol, starts to crystallize already from
the hot reaction mixture. The isolation of E2 crystals during the re-
action process displaces logically the E14E2 transformation to-
wards E2 as well as the entire synthesis route from 1 to E.
The formation of the a,b-unsaturated ketone F can be tentatively
explained by the gradual hydrolysis of E1 and E2 caused by the
presence of water, generated in situ during the formation of B and C
(Scheme 1). However, studying the literature, one might oppose
that the synthesis to the
according to an other wide-spread mechanism: following the
classic aldol reaction, a related -hydroxyketone G would be the
a,b-unsaturated ketone F can proceed
3.4. NMR study of rifamycin 6 (type F): properties of the
substituent at the C-3 atom (Fig. 2, Table 3)
b
product of a reaction between ketone and starting aldehyde 1 (see
Scheme 2 for overview).28 Albeit this compound G was not directly
observed in the reaction mixture, its formation cannot be com-
In the 13C NMR spectrum, the signal at 130.0 was assigned to C-10
whereas proton 10-H was found at 7.34 ppm as a doublet. In the
1H,13C HMBC spectrum a 10-H/C-2 and 10-H/C-30 correlations were
pletely ruled out. The
a,b-unsaturated ketone F (G/F trans-
formation, Scheme 2) would readily form via elimination of a water
molecule. On the other hand, speaking against this aldol-like con-
densation is the fact that ketone F is formed relatively late in the
reaction course and that an increase of its concentration is clearly
accompanied (TLC) by a decrease of the concentration of imine E.
This supports the E to F route.
1
found. In the H,1H COSY spectrum a clear 10-H/20-H, correlation
signal was found, and the high value of coupling constant
0
0
0
0
J1 ,2 ¼16.0 Hz proves a trans configuration of protons 1 -H and 2 -H.
The characteristic low-field signal at 200.0 ppmwas attributedtothe
carbonyl carbon C-30 as well as the methylene C-40 to the signal at
49.8 ppm. In the 1H NMR spectrum the magnetically non-equivalent
methylene groups 40-Ha and 40-Hb give multiplets at 2.44 and
2.55 ppm. The 13C signal at 24.7 ppm was assigned to C-50 while the
related proton 50-H could be found as a multiplet at 2.03 ppm,
overlapping with signals of 30-H and 36-H in the 1H spectrum. The
signals at 22.4 and 22.3 ppm were assigned to methylene groups C-
60a and C-60b in the 13C NMR spectrum whilst the 1H-signals of these
groups overlap at 0.84 ppm (6a0 -3H and 60b-3H as doublets). Contrary
to the situation found in 2, the rotation around the C-40eC-50 bond is
only slightly inhibited by the change of the alkyl substituents around
C40eC50, the steric hindrance being mainly limited to an interaction
involving isopropyl fragment and ketone group.
Considering other mechanistical possibilities, another reaction
can be also taken into account (Scheme 2): The
formed from imine B and the starting ketone, can react with the
starting amine to yield -aminoimine D. This compound consecu-
tively abstracts an amine and a water molecule to produce com-
pound (1/B/H/D/E/F reaction route). However
b-aminoketone H,
b
F
confronting this mechanism to the findings of the TLC analysis, it is
quite obvious that the proposed intermediates H and D have to be
observed as distinct spots on the chromatograms:
If the hypothetical compound H was formed, it should be most
likely detectable, since a very rapid occurrence of the H/D process
is doubtful (an analogous 1/B reaction takes 1e4 h to be com-
pleted). In the reaction system at hand no spot is observed that
could be assigned to H. Moreover, H would convert directly into the
3.5. Selected elements of NMR analysis of the rifamycin 6
ansa-frame
a,b-unsaturated ketone F, splitting off an amine molecule instead of
forming E (B/H/F transformation). This again widely diverges
from the TLC findings observed during the conversion of B to F.
Literature reports have been found describing pathways analo-
gous to the main synthetic route proposed in Scheme 1. For in-
stance the acid-catalysed, aldol type condensation of two imine
In the low-field part of the 1H NMR spectrum a broad signal
characteristic of a mobile proton was found at 13.04 ppm, probably
8-OH, whereas a singlet at 8.92 ppm, showing H/C long range
coupling with C-1, C-2 and C-15 was assigned to the proton of the
amide group present in the ansa chain. The broad bulge around
8.0 ppm noticed in the baseline of the 1H NMR spectrum is most
likely due to rapid exchange phenomena involving four mobile
protons: 1-OH, 4-OH, 21-OH and 23-OH (the latter two signals and
the baseline hump disappeared after deuterium exchange via D2O
addition).
molecules having protons in
-aminoimines, these compounds losing an amine to form the
corresponding
-unsaturated imine.29e31
a-position of the imino group to form
b
a,b
5. Conclusions
In the reaction of 3-formylrifamycin SV (1) with primary alkyl-
amines and ketones displaying a R1eCH2eCO-R2 structure, where
R1]H or alkyl and R2]alkyl, aryl (Scheme 1), a new group of rifa-
4. Mechanism proposal for the reaction of 1 with primary
amines and ketones
mycin derivatives, displaying a,b-unsaturated imines substituents
As summarized in Scheme 1, a chain of successive reactions is
initiated by the condensation of 3-formylrifamycins SV (1) with an
(E1 and E2) at carbon C-3 could be isolated and fully characterized
via NMR and MS-methods. These compounds showed a tendency to
reversibly isomerise in organic solvents and, in the presence of
water, to rapidly hydrolyse. Only some derivatives presenting
alkylamine catalyzed by acetic acid. The so-formed
a-aminoalcohol
A affords, after abstraction of a water molecule, the violet-blue

5932
K. Bujnowski et al. / Tetrahedron 68 (2012) 5925e5934
Table 3
13C and 1H NMR data of 6 in (D6)DMSO
Atom C
C-1
d
Atom H
(amide)eH
d
Multipl.
s
JH,H
1H,1H COSY correlations
1H,13C HMBC correlations
C-1, C-2, C-15
N
8.92
147.5
b
1-OH
C-2
C-3
C-4
118.4a
118.5a
148.3
b
4-OH
C-5
C-6
C-7
C-8
99.8
172.1
102.1
181.7
8-OH
13.04 br
C-9
114.8
118.5a
186.4
108.7
22.0
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
3 13-H
3 14-H
1.67
1.94
s
C-11, C-12
C-6, C-8
7.5
sa
168.2
131.7a
131.8a
126.4
138.8
37.7
17-H
18-H
19-H
20-H
21-H
21-OH
22-H
23-H
23-OH
24-H
6.31
6.83
6.03
2.21
d
(17,18)¼11.0
(18,19)¼16.0
(19,20)¼7.4
18-H, 3 30-H lr.
17-H, 19-H
18-H, 20-H
19-H, 21-H, 3 31-H
20-H
dd
dd
ma
m
C-17
72.8
3.67
b
C-22
C-23
38.1
75.8
1.24
2.76
m
m
23-H, 3 32-H
22-H, 24-H
b
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
32.3
73.2
40.0a
76.2
117.7
142.9
20.3
17.3
9.1
1.57
5.07
1.03
3.24
4.89
6.25
1.96
0.77
0.42
0.87
ꢁ0.40
m
dd
ma
d
23-H, 3 33-H
26-H
25-H
26-H
27-H
28-H
(25,26)¼10.8
C-35
25-H, 3 34-H
28-H, 29-H lr.
27-H, 29-H
27-H lr., 28-H
17-H lr.
20-H
22-H
24-H
26-H
(27,28)¼8.2
dd
d
(28,29)¼12.8
29-H
C-12, C-27, C-28
C-15, C-16
C-19, C-20, C-21
C-22, C-23, C-25
C-21, C-23, C-24
C-26, C-27
3 30-H
3 31-H
3 32-H
3 33-H
3 34-H
sa
d
(31,20)¼6.8
(32,22)¼6.8
(33,24)¼6.8
(34,26)¼6.8
d
10.7
8.9
da
d
169.3
20.6
55.6
130.0
136.5
200.0
49.8
3 36-H
3 37-H
10-H
1.95
2.88
7.34
7.58
sa
s
d
d
C-35
C-27
C-2, C-30
C-3, C-30
(10,20)¼16.0
20-H
10-H
C-20
20-H
C-30
C-40
40-Ha
40-Hb
50-H
2.44
2.25
2.03
0.84
0.84
dda
dda
ma
da
(40a40b)¼12.8
(40a,50)¼6.8
(5040b)¼6.8
40-Hb, 50-H
C-50, C-60a, C-60b
C-50, C-60a, C-60b
C-60a, C-60b
40-Ha, 50-H
C-50
C-60a
C-60b
24.7
40-Ha, 40-Hb, 3 60a-H, 3 6b0 -H
22.4a
22.3a
3 60a-H
3 60b-H
50-H
50-H
C-50, C-60b
da
C-50, C-60a
Abbreviations: s: singlet, d: doublet, m: multiplet, br: broad, lr: long range correlation.
a
This signal overlapped with another signal.
Shared signaldbroad bulge of the spectrum baseline with a maximum at ca. 8.0 ppm.
b
definite structural characteristics could be isolated in a crystalline,
stable form: the compounds displaying substituents R2]CH2-A,
with A]alkyl, R2]aryl, and with R3]H. Representativefor this class
of compounds, two type-E1 and two type-E2 rifamycins as well as
the tested compounds showed a marked anti-tuberculous activity
and activity against MOTT, although not higher than the activity of
the reference materials.32
Imines E1 (generallydblue, crystallizing from chloroform) and
E2 (generallyddark blue, crystallizing from methanol) are most
probably geometric isomers, differing in the configuration (syn and
anti) of substituents R2 and R at the imine bond (Scheme 1). The
observed shifts in 13C NMR spectra of the signals attributed to
a connected hydrolysis product, a
a,b-unsaturated ketone, were
reported in this contribution. The general synthetic method to ob-
tain these N-substituted 3-(30-imino)alkylidenylrifamycins SV is the
subject of an ongoing patent application. Some of the new N-
substituted 3-(30-imino) alkylidenylrifamycins SV, among others
compound 2, were tested in vitro for tuberculostatic activity against
different strains of Mycobacterium and compared to the main rifa-
mycin antibiotics: rifampicin (RMP) and rifabutin (RBT). For the test
standard strain of Mycobacterium tuberculosisdM. tbc H37Rv and M.
tbc Bovis were used, as well as different strains of MOTT (Mycobac-
teria Other Than M. Tuberculosis) sensitive or resistant to RMP and
RBT. In spite of the already mentioned trend towards hydrolysis, all
C(a)eC]Ne, e.g., for 4 of type E2 in CDCl3 solution, correlate with
the gradual isomerisation to form E1 and suggests that these sub-
stituents have an anti arrangement in isomers E1 and a syn one in
isomer E2.
Along these lines, it could be noticed that structure E1 generally
shows higher stability in solution, probably due an energetically
more favourable spatial arrangement of the substituents at the
imine C]N bond.

K. Bujnowski et al. / Tetrahedron 68 (2012) 5925e5934
5933
remaining signal of the non-deuteriated part of the solvent
(2.50 ppm) and the C-D characteristic coupling pattern of the
deuteriated solvent in 13C (39.5 ppm), when necessary a supple-
mentary calibration with TMS as internal standard has been done.
The 2D, short- and long range correlated C,H- spectra (gHSQC and
gHMBC, respectively) were recorded using standard pulse se-
quences with z-gradients, as provided by Varian with the VNMR
6.1C control and processing software. Mass spectra (MS) and high-
resolution mass spectra (HRMS) were recorded by employing
a Mariner (PerSeptive Biosystems) mass scectrometer using the
electro spray ionization (ESI) technique and are reported as m/e
(relative intensity). Accurate masses are reported for the molecular
ion (Mþ1) or a suitable fragment ion. For this analysis the samples
were dissolved in CH3OH. IR spectra were recorded on a Per-
kineElmer PE-577 instrument using KBr pellets. The new rifa-
mycin derivatives have no definite melting pointsdthey do not
melt (up to 300 ꢀC) and slowly decompose at temperatures over
160 ꢀC.9
O
O
O
15
15
NH
15
NH
R
NH2
NH
- H2O
2
3
R = alkyl, H
2
3
2
3
H
N
N
O
C
H
AcOH (cat.)
R
R
OH
3-formylrifamycin SV (1)
(A)
(B)
R2
R1
R2
R1 = alkyl, H
2 = alkyl, aryl
R1
R
NH2
O
R
O
(cat.)
R
NH2
(cat.)
O
15
O
O
15
NH
NH
15
2
3
NH
(F)
2
3
2
3
H
-
R
NH2
- H2O
N
OH
R2
R
R2
R2
AcOH,R NH2
(cat.)
AcOH, R NH2
(cat.)
R1
R1
R1
O
O
O
(G)
(H)
6.2. General synthetic procedure for a,b-unsaturated imines E
R
NH2
H2O
O
-
R
NH2
- H2O
AcOH (cat.)
1.6 g of 3-formylrifamycin SV (2 mmol) was added to a solution
containing: 15.0 ml of methanol, 0.6 g of acetic acid, 5e10 mmol of
amine and 5e20 mmol of ketone. The system was stirred at
38e45 ꢀC, chromatographically (TLC) monitoring the reaction
progress. (The red spot of 3-formylrifamycin SV gradually disap-
pears whereas two products spots appear and then enlargeda blue
one for type E1 imine and a navy blue one for imine E2, with Rf
E1<Rf E2).
O
15
15
NH
NH
2
3
2
3
-
R
H
NH2
N
R
R2
AcOH,R NH2
(cat.)
R2
R1
R1
N
N
R
R
(E)
(D)
After disappearance of 3-formylrifamycin SV or reaction in-
hibition via hydrolysis, only an increase in intensity of the red spot
of ketone Fd(hydrolysis of E; RfF<RfE1) was observed, the process
was stopped by cooling down the system to room temperaure.
Reaction time: 0.5e4 h.
Scheme 2. Alternative routes of reaction of 1 with primary amines and ketones.
Due to a general propensity to hydrolysis, the new rifamycin SV
derivatives, exhibiting a,b-unsaturated iminic substituents at C-3,
are not directly usable as potential medicines, but they are defi-
nitely valuable reactive derivatives for further rifamycin
modifications.
A) When the a,b-unsaturated imines crystallized from methanol
in the form of type E2 isomer, e.g., 3 and 4, the post-reaction
mixture was left for 16 h at w5 ꢀC. The crystalline dark blue
product was filtered off, washed with cold (w5 ꢀC) methanol,
and then with hexane followed by drying under vacuum.
In the reaction system studied here, imines E play a role as in-
termediates in the synthesis of rifamycins displaying
a,b-un-
saturated ketones at C-3 (compound F). The general mechanism of
formation, proposed in Scheme 1, is different from the one de-
scribed for classical aldol reactions, the key-step in our case being
the formation of imino-derivatives, B and C.
The condensation of B- and C-type imines, formed in situ seems
to be a promising, although hitherto rarely employed route to form
B) When a,b-usaturated imines did not crystallize from methanol
(this concerns derivatives of 4-methyl-2-pentanone (isobutyl-
methyl ketone) and 2,4-dimethyl-6-heptanone), 40 ml of
chloroform and 60 ml of water were added to the post-reaction
mixture. After mixing the reaction mixture at 40e45 ꢀC, the
chloroform phase was separated and washed with 120 ml of
water. The organic phase was then concentrated to a volume of
10e20 ml, and left for 16 h at w5 ꢀC. The crystalline blue
a
,
b
-unsaturated imines or, via further hydrolysis, to form the re-
lated -unsaturated ketones.
a,b
productda,b-unsaturated imine as E1 isomerdwas filtered off,
6. Experimental part
6.1. General
washed with cold (w5 ꢀC) chloroform, then hexane and finally
dried under vacuum.
Available commercial reactants were used without additional
purification: 3-formylrifamycin SV (1) (provided by Pol-
faeTarchomin), ketones, primary amines, carboxylic or sulfonic
acids (alldsynthesis grade purity: >98%) as well as solvents
(alld99% min.). TLC chromatograms were developed on aluminium
plates covered with silicagel 0.2 mm 60 F254, using CHCl3/MeOH
(9:1 v/v) as the mobile phase.
6.3. Synthesis of the a,b-unsaturated imines 2, 3, 4, 5
Synthesis of 3-(30-Isopropylimino-50-methyl)hexenylrifamycin
SV (2) (of type E1): 1.6 g of 3-formylrifamycin SV (2 mmol) was
added to a solution containing 15.0 ml of methanol, 0.6 g of acetic
acid, 0.6 g (w10 mmol) of isopropylamine and 2.0 g (w20 mmol) of
4-methyl-2-pentanone. The system was stirred at 40e45 ꢀC for 4 h.
The crystalline blue product was separated according to 6.2-B. 1,2 g
of 2 was obtained. Yield 70.6%. See Table 2 for 13C and 1H NMR
spectroscopic data. MS (ESI): m/z (%): 849.5 (100.0) [MþH]þ, 871.4
(91.7) [MþNa]þ, 812.4 (20.8) [MþNaꢁC3H7NH2]þ, 790.4 (7.1)
[MþHꢁC3H7NH2]þ. HRMS calcd for [MþNa]þ C47H64N2O12Na
The 1H and 13C NMR were recorded operating at 399.91 and
100.56 MHz, respectively, by means of a Varian InovaUnity 400
spectrometer equipped with an Oxford Magnet (9,4T). All samples
were dissolved in ca. 0.5 ml of DMSO-d6. Chemical shifts are
reported in parts per million, and J-coupling constants are
expressed in Hertz. The calibration was performed using the

5934
K. Bujnowski et al. / Tetrahedron 68 (2012) 5925e5934
871.4351; found 871.4376 (error: 2.7 ppm). IR: 3400, 3240, 2960,
1700, 1600, 1535, 1470 cmꢁ1
(error: 2.6935 ppm). IR: 3408 (br.), 2968, 1723, 1594, 1372,
1240 cmꢁ1
.
.
Synthesis of 3-(50-Methyl-30-propylimino) hexenylrifamycin SV
(3) (of type E1): 1.6 g of 3-formylrifamycin SV (2 mmol) was added to
a solution containing: 15.0 ml of methanol, 0.6 g of acetic acid, 0.6 g
(w10 mmol) of propylamine and 2.0 g (w20 mmol) of 4-methyl-2-
pentanone. The system was stirred at 40e45 ꢀC for 4 h, The crystal-
line blue product was separated according to 5.2-B. 1,05 g of 3 was
obtained. Yield 61.8%. See Supplementary data for 13C and 1H NMR
spectroscopic data. MS (ESI): m/z (%): 871.5 (100.0) [MþNa]þ, 812.4
(71.5) [MþNaꢁC3H7NH2]þ, 849.5 (45.5) [MþH]þ, 790.4 (15.2)
[MþHꢁC3H7NH2]þ. HRMS calcd for [MþNa]þ C47H64N2O12Na
871.4351;found871.4362(error:1.2ppm). IR:3409, 3246, 2968,1704,
Acknowledgements
The above research project was financially supported by War-
saw University of Technology. The authors kindly thank Mr. Norbert
Langwald and Ms. Renata Przedpe1ska for the editorial assistance,
and Mr. Gilbert Zwick for recording the MS-ESI spectra.
Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2012.04.071. These data in-
clude MOL files and InChiKeys of the most important compounds
described in this article.
1596, 1472, 1247 cmꢁ1
.
Synthesis of 3-(30-Cyclohexylimino)octenylrifamycin SV (4) (of
type E2): 1.6 g of 3-formylrifamycin SV (2 mmol) was added to
a solution containing: 15.0 ml of methanol, 0.6 g of acetic acid,
1.0 g (w10 mmol) of cyclohexylamine and 2.3 g (w20 mmol) of 2-
heptanone. The system was stirred at w45 ꢀC for 2/3 h. The
crystalline dark blue product was separated according to 6.2-A.
0.92 g of 4 was obtained. Yield 51.0%. See Supplementary data for
13C and 1H NMR spectroscopic data. MS (ESI): m/z (%): 925.5
(100.0) [MþNa]þ, 908.5 (38.1) [MþH]þ, 826.4 (32.7) [MþNa-
C6H11NH2]þ. HRMS calcd for [MþNa]þ C51H70N2O12Na 925.4821;
found 925.4830 (error: 1.0 ppm). IR: 3458 (br), 2936, 2864, 1716,
References and notes
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Synthesis of 3-(30-Isopropylimino)nonenylrifamycin SV (5) (of
type E2): 1.6 g of 3-formylrifamycin SV (2 mmol) was added to a so-
lution containing: 15.0 ml of methanol, 0.6 g of acetic acid, 0.6 g
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[MþNaꢁC3H7NH2]þ, 877.5 (37.0) [MþH]þ HRMS calcd for [MþNa]þ
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.
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