Investigation on Structure-Bioactivity Relationship and
Determination of the Absolute Configuration of Natural Products
Der Fakultät für Naturwissenschaften
Department Chemie
der Universität Paderborn
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
Dr. rer. nat.
genehmigte Dissertation
von
Brigitta Elsässer
aus Nyíregyháza, (Ungarn)
Paderborn 2004
Eingereicht am: 26.10.2004
Mündliche Prüfung: 10.12.2004
Referent: Prof. Dr. Karsten Krohn
Korreferent: Prof. Dr. Sándor Antus
Die vorliegende Arbeit wurde in der Zeit von April 2001 bis September 2004 im Fach
Organische Chemie der Fakultät Naturwissenschaften der Universität Paderborn unter
Anleitung von Herrn Prof. Dr. Karsten Krohn angefertigt.
Herrn Prof. Dr. Karsten Krohn danke ich herzlich für die Möglichkeit dieses interessante und
vielseitige Thema zu bearbeiten, sowie für seine Unterstützung durch zahlreiche anregende
Diskussionen.
Bei Herrn Prof. Sándor Antus möchte ich mich für die Übernahme des Korreferates bedanken.
Weiterhin gilt mein Dank:
Herrn Prof. Dr. G. Fels für die vielen anregenden Diskussionen.
Herrn Prof. Dr. H. Marsmann für die Messung von NMR-Spektren.
Herrn Dr. Tibor Kurtán für die Messung und Diskussion der CD-Spektren.
Herrn Dr. M. Morr für seine Hilfe bei der Herstellung des Benzamidribosid-Adenin-
Dinukleotids.
Herrn Prof. Dr. H. N. Jayaram für die rasche Durchführung der biologischen Testungen.
Herrn Ulrich Flörke für die Röntgenstrukturanalysen.
Herrn Dr. Jürgen Vitz, Herrn Dr. Klaus Steingröver, Herrn Dietmar Gehle, Herrn Ivan
Shuklov und den anderen Mitarbeiterinnen und Mitarbeitern der Organischen Chemie für das
kollegiale und freundliche Arbeitsklima.
Herrn Alexander Janzen und Herrn Gábor Májer für ihre Mitwirkung bei einigen
Synthesestufen.
Frau Mariola Zukowski für die Messung der Massenspektren, Herrn PD. Dr. Hans Egold für
die 500 MHz NMR Spektren.
Frau Boripont Manmont und Danielle Walter für das Korrekturlesen.
Mein ganz persönlicher Dank gilt meinem Mann, Robert, der immer an mich geglaubt hat.
Ich bedanke mich bei der DAAD und bei der Kommission für Forschung und
Wissenschaftlicher Nachwuchs für die finanzielle Unterstützung.
“The way to succeed is to keep one´s courage and patience,
and to work on energetically.”
(Vincent van Gogh, 1853-1890)
Table of Contents
1 INTRODUCTION............................................................................................................................... 1
1.1 CANCER ................................................................................................................................................. 1
1.1.1 Treatment ........................................................................................................................................ 2
1.2 APOPTOSIS ............................................................................................................................................. 3
1.3 AIMS AND SCOPES ................................................................................................................................. 5
2 SYNTHESIS OF BENZAMIDE RIBOSIDE DERIVATIVES WITH ANTITUMOR
ACTIVITY............................................................................................................................................................. 7
2.1 INTRODUCTION ...................................................................................................................................... 7
2.2 MECHANISM OF ACTION OF BENZAMIDE RIBOSIDE ............................................................................... 8
2.3 SYNTHESIS OF BENZAMIDE RIBOSIDE .................................................................................................... 8
2.3.1 Synthesis of C-glycosides ................................................................................................................ 8
2.3.2 Preparation of 2,3,5-tri-O-benzyl-
γ
-ribonolactone (2-7)................................................................ 9
2.3.3 Coupling of 1,4–Ribonolactone (2-7) with organometallic reagent 2-11 ..................................... 11
2.3.4 Cleavage of the oxazoline group and transformation to benzamide riboside (2-2) ...................... 12
2.4 SYNTHESIS OF BENZAMIDE RIBOSED ANALOGUES .............................................................................. 14
2.4.1 Preparation of 3-deoxy sugar derivative (2-32)............................................................................ 14
2.4.2 Preparation of derivatives with different substituents on the aromatic ring................................. 17
2.5 INVESTIGATION TOWARDS THE SYNTHESIS OF 2-DEOXY- AND 2,3-DIDEOXY-BENZAMIDE RIBOSIDE .. 20
2.5.1 Preparation of 2,3-dideoxy-5-benzyl-ribonolactone (2-46) .......................................................... 20
2.5.2 Preparation of 2-deoxy-3,5-dibenzyl-ribonolactone (2-52) .......................................................... 22
2.5.3 Investigation on the coupling of deoxy-lactones 2-46 and 2-52 with lithiated oxazoline 2-11 ..... 23
2.6 BIOLOGICAL TEST RESULTS.................................................................................................................. 24
3 SYNTHESIS OF BENZAMIDE RIBOSIDE ADENINE DINUCLEOTIDE............................... 26
3.1 INTRODUCTION .................................................................................................................................... 26
3.2 SYNTHESIS OF BENZAMIDE RIBOSIDE ADENINE DINUCLEOTIDE (3-5) ................................................... 27
3.3 ENZYM-SUBSTRATE COMPLEX STUDIES ............................................................................................... 28
4 SYNTHESIS AND STRUCTURE-ACTIVITY RELATIONSHIP OF ANTIFUNGAL
CONIOTHYRIOMYCIN ANALOGUES......................................................................................................... 29
4.1 INTRODUCTION .................................................................................................................................... 29
4.2 DEVELOPING A NEW METHOD FOR THE SYNTHESIS OF MIXED IMIDES................................................... 30
4.3 SYNTHESIS OF CONIOTHYRIOMYCIN ANALOGUES – VARIATION OF THE SUBSTITUTION PATTERN OF THE
BENZYL-RING....................................................................................................................................... 31
4.4 SYNTHESIS OF CONIOTHYRIOMYCIN ANALOGUES – CHANGING THE DEGREE OF SATURATION, AND
HYDROPHOPBICITY IN THE FUMARIC ESTER SIDE-CHAIN ...................................................................... 32
4.5 SYNTHESIS OF CONIOTHYRIOMYCIN ANALOGUES - REPLACEMENT OF CARBON BY NITROGEN, OXYGEN
OR SULFUR IN THE MIDDLE PART OF THE MOLECULE ............................................................................ 33
4.6 BIOLOGICAL STUDIES .......................................................................................................................... 34
5 STRUCTURE-ACTIVITY RELATIONSHIPS IN ALLERGIC CONTACT DERMATITIS .. 36
5.1 INTRODUCTION .................................................................................................................................... 36
5.1.1 The investigated phenanthrene-quinones...................................................................................... 38
5.2 SENSITIZATION .................................................................................................................................... 40
5.3 SENSITIZATION TEST RESULTS.............................................................................................................. 41
5.4 QUANTUM MECHANICAL CALCULATIONS............................................................................................. 44
6 DETERMINATION OF THE ABSOLUTE CONFIGURATION BY QUANTUM CHEMICAL
CALCULATION OF CD SPECTRA................................................................................................................ 47
6.1 INTRODUCTION .................................................................................................................................... 47
6.2 THEORETICAL PRINCIPLES - CALCULATION OF CD SPECTRA............................................................... 48
6.3 DETERMINATION OF THE ABSOLUTE CONFIGURATION OF AN ERGOCHROME ........................................ 51
6.3.1 Atropismerism ............................................................................................................................... 54
6.3.2 Determination of rotation barrier of ergochrome (6-1)................................................................ 55
6.4 CALCULATION OF THE ABSOLUTE CONFIGURATION OF EPIEUDESMIN (6-4).......................................... 58
6.5 DETERMINATION OF THE ABSOLUTE CONFIGURATION OF A XYLOKETAL (6-5) .................................... 60
6.6 DETERMINATION OF THE ABSOLUTE CONFIGURATION OF ASCOCHIN (6-6) .......................................... 62
6.7 DETERMINATION OF THE ABSOLUTE CONFIGURATION OF A METABOLIC PRODUCT OF PHOMOPSIS
OBLONGA (6-8) USING UV-CORRECTION............................................................................................... 66
6.8 DETERMINATION OF ABSOLUTE CONFIGURATION OF PLUMERICINE (6-9) AND PLUMENOSIDE (6-10)..69
7 SUMMARY ....................................................................................................................................... 72
8 EXPERIMENTAL PART ................................................................................................................ 74
8.1 INSTRUMENTATION.............................................................................................................................. 74
8.2 EXPERIMENTAL PART TO CHAPTER 2................................................................................................... 75
8.3 EXPERIMENTAL PART TO CHAPTER 3................................................................................................. 100
8.4 EXPERIMENTAL PART TO CHAPTER 4. ................................................................................................ 103
9 ABBREVIATIONS ......................................................................................................................... 118
10 LITERATURE ................................................................................................................................ 119
Introduction
1
1 Introduction
The unambiguous knowledge of the stereo structure of biologically active synthetic and
natural products is an important prerequisite for structure-activity relationship (SAR)
investigation. For this reason this thesis involves both the studies on structure-activity
relationship and the determination of the absolute stereo structure.
Other applications, such as targeted drug design and delivery can also profit from the
investigation on structure-activity relationship. SAR is a means by which the effect of a drug
or toxic chemical on an animal, plant or the environment can be related to its molecular
structure. This type of relationship may be assessed by considering a series of molecules and
making gradual changes to them, noting the effect upon their biological activity of each
change. In these investigations the first task is to discover which part of the molecule is
responsible for the biological activity. Using this information, the next step is to modify the
other part of the compound to reach the required activity.
1.1 Cancer
The most important investigation on structure-bioactivity relationship of this thesis is the
synthesis of benzamide riboside derivatives. Benzamide riboside (2-2) exhibits potent
antitumor activity against several human cancer cells.[1,2] Therefore 2-2 might be a promising
antitumor agent in the cancer therapy.
Although there are many kinds of cancer, they all start because of out-of-control growth of
abnormal cells. Cancer develops when cells in a part of the body begin to grow out of control
because of damage to DNA. Most of the time when DNA gets injured, either the cell dies or it
is able to repair the DNA. In cancer cells, the damaged DNA is not repaired. People can
inherit damaged DNA, which accounts for inherited development of cancers.[3,4] Many times
though, a person’s DNA can be harmed by exposure to carcinogens in the environment, like
smoking. Cancer usually forms as a tumor. Some cancers, like leukemia, do not form tumors.
Instead, these cancer cells involve the blood and blood-forming organs, and circulate through
other tissues where they grow. Cancer cells often spread to other parts of the body where they
begin to grow and replace normal tissue. This process, called metastasis, occurs as the cancer
cells get into the bloodstream or lymph vessels of our body. Tumors can be classified into two
groups:
Introduction
2
• Benign tumors are not cancerous. They can often be removed and, in most cases, they
do not come back. Cells from benign tumors do not spread to other parts of the body.
Most importantly, benign tumors are rarely a threat to life.
• Malignant tumors are cancerous. Cells in these tumors are abnormal and divide
uncontrollably and disorderly. They can invade and damage nearby tissues and organs.
Also, cancer cells can break away from a malignant tumor and enter the bloodstream
or the lymphatic system. That is how cancer spreads from the original cancer site to
form new tumors in other organs.
Leukemia and lymphoma are cancers that arise in blood-forming cells. The abnormal cells
circulate in the bloodstream and lymphatic system.[5] They can also invade (infiltrate) body
organs and form tumors.
1.1.1 Treatment
Treatment for cancer depends on the type of cancer; the size, location, and stage of the
disease; the person's general health; and other factors. Treatment for cancer can be either local
or systemic. Local treatments affect cancer cells in the tumor and the surrounding area.
Systemic treatments travel through the bloodstream, reaching cancer cells all over the body.
Surgery and radiation therapy are types of local treatment. Chemotherapy, hormone therapy,
and biological therapy are examples of systemic treatment.[6,7]
It is hard to protect healthy cells from the harmful effects of cancer treatment. This is because
treatment does damage healthy cells and tissues. Moreover, it often causes side effects. The
side effects of cancer treatment depend mainly on the type and extent of the treatment. Also,
the effects may not be the same for each person.
Chemotherapy is the use of drugs to kill cancer cells. It may be the only kind of treatment a
patient needs, or it may be combined with other forms of treatment. Neoadjuvant
chemotherapy refers to drugs given before surgery to shrink a tumor; adjuvant chemotherapy
refers to drugs given after surgery to help prevent the cancer from recurring. Chemotherapy
also may be used (alone or along with other forms of treatment) to relieve symptoms of the
disease. Sometimes the anticancer drugs are given in other ways. For example, in an approach
called intraperitoneal chemotherapy, anticancer drugs are placed directly into the abdomen
through a catheter. To reach cancer cells in the central nervous system (CNS), the patient may
receive intrathecal chemotherapy. In this type of treatment, the anticancer drugs enter the
Introduction
3
cerebrospinal fluid through a needle placed in the spinal column or a device placed under the
scalp.
The side effects of chemotherapy depend mainly on the drugs and the doses the patient
receives. Generally, anticancer drugs affect cells that divide rapidly. In addition to cancer
cells, blood cells, which fight infection, help the blood to clot, and carry oxygen to all parts of
the body that are affected. When blood cells are affected, patients are more likely to get
infections, may bruise or bleed easily, and may feel unusually weak and very tired. Rapidly
dividing cells in hair roots and cells that line the digestive tract may also be affected. As a
result, loss of hair, poor appetite, nausea and vomiting, diarrhea, or mouth and lip sores are
possible side effects that may arise.
Inhibiting cancer involves more than just locating the right molecule. Most cancers engage
multiple growth factor, angiogenic, cell cycle, and apoptosis pathways. Frequently, redundant
pathways exist, so that as drugs shut one pathway down another pathway takes over. This is
one way that cancer becomes resistant to targeted agents. Early stage tumors tend to secrete a
small number of pro-angiogenic factors, whereas late stage tumors secrete a larger number of
pro-angiogenic factors.
1.2 Apoptosis
Until several years ago, cell death has been regarded as an event that is in principle negative
for the organism. The fact that cell death possesses an important regulating function during
development of the organism and it represents a central part of the life, was a surprising
discovery. Programmed cell death, the apoptosis, is a suicide program of the cell, which
completely eliminates the cell within a few hours. This phenomenon has already been well-
known for decades. However, in 1972, after the detailed description of the morphologic
changes during apoptosis[8], the physiological cell elimination was recognized as an
independent and genetically controlled form of cell death, and the term Apoptosis was given.
The term apoptosis (apo = off, away; ptosis = lowering) originates from Greek and describes
the drop of leaves in autumn. Five years later the first genes, which are responsible for the
apoptosis, were discovered in the thread worm “Caenorhabditis elegans”. The genes were
found by mutations, which lead to the disturbance of the apoptosis during the growth.[9] In
1990 researchers discovered that a tumor production gene in case of overexpression prevents
apoptosis.[10] Thus the apoptosis had become one of the most attractive new research fields
Introduction
4
with constantly rising publication numbers. So far, this development has appeared in over
50000 scientific publications relating to this topic.
Apoptosis is an important possibility for multi-cell organism to organize themselves. It plays
an important role in directing apoptotic death of certain cells during the development of the
embryo. The shaping of body and organs of the embryo during the development also takes
place via apoptosis; for example the skins between toes and fingers are removed by apoptosis
during the embryonic growth. Nerve cells are produced in excess during the embryonic
development and as soon as they do not have contact with the neighboring neurons its level is
lowered through apoptosis to 40-85%. Without contact to the neighbors the nerve growth
factors (NGF) or ChAT (choline acetyltransferase) development factors (CDF)[11] are missing.
Later on the central nerve-system shows only a small cell-elimination-rate. Each diseased
change in the genetic material of the cells would be passed on inevitably to the
descendants.[12]
A cell begins the apoptosis program, if internal or external signals command the self
destruction.
Figure 1.1: Pathway of apoptosis
Introduction
5
Each cell of our body is in constant contact to its neighboring cells by chemical messenger
materials (e.g. growth factor NGF or Interleukin-2), which is bound to the receptors of the
cell. This contact is extracted and usually leads to the release of apoptosis.
High doses of UV or X-ray radiation can also lead to damage of the genetic material in the
cell. These cells then have the choice between repairs of smaller DNA damage, or if the
damage can not be repaired any more then apoptosis. In case of doubt, the cell kills itself, in
order not to degenerate.
Cancer cells differ from other cells in particular by the fact that they proliferate unresisted. A
change in the genetic code made these cells immortal. The mutated genes are often dealing
with apoptosis-regulation. In 50 % of the human tumors genes, which the Tumor-suppressor-
protein P53 code, are inactive. In the case of some kinds of cancer, high concentrations of
apoptosis inhibitors Bcl-2 were found. The aim of the medical research is to find a medicine
against such apoptosis resistant cancer cells.
Depending on cell type and elicitor, the apoptosis can be introduced by three different signal
paths. The common goal of the different apoptosis activating way is the initiation of Caspase
cascade, which introduces irreversible cell death.[13]
1.3 Aims and Scopes
In order to improve the biological activity of some natural and synthetic compounds, several
of their derivatives had to be synthesized and tested. Through systematic changing of the
different moieties and/or substitution patterns of the molecules, and performing biological
tests it can be established which part of the molecule is responsible for the bioactivity. This
thesis consists of five topics, from which four are concentrating on structure-bioactivity
relationship investigation. The fifth deals with the determination of the absolute configuration
of natural products.
(1) The first project concentrates on the synthesis of antitumor benzamide riboside
derivatives in hope of improving a higher biological selectivity against cancer cells.
O
R
1
R
1
HO
R
2
R
2
: CONH
2
, OMe, or OH, F, H
R
1
: OH, or H
6
(2) The second part involves the preparation of NAD analogous BAD in order to investigate
the enzyme-substrate complex with ubichinon. X-ray crystallographic study could
provide information about the active site of reductases.
ON
CONH
2
HO OH
OPO
O
OH
P
OH
O
O
O
N
N
N
NH
2
N
OHHO
BAD
(3) The third topic describes the synthesis and biological study of antifungal
coniothyriomycin analogues to find stable derivatives with high activity against plant
fungi.
H
2
CNOR
OO
HO
R
1
R
1
: Variation of the substitution pattern of the aromatic ring
Replacement of the phenylacetic acid with benzoic acid
Variation of the degree of saturation in the side chain
R: Changing the hydrophobicity of the alcohol component
Replacement of the imide function to hydrazide, thio, etc. derivatives
(4) The fourth project is to prove a so far unknown correlation between the molecule orbital
coefficients (LUMO) and the biological activity on the example of several
phenanthrenequinones.
O
OR
R
R
R
R
R R
R: H or OMe
(5) The last topic focuses on the determination of the absolute structure of natural products
using a quantum mechanical approach. By calculating the CD-spectra of compounds and
comparing them with that of the experimental ones, the absolute configuration of the
given molecule can be elucidated.
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
7
2 Synthesis of Benzamide Riboside Derivatives with Antitumor
Activity
2.1 Introduction
Benzamide riboside, a recently discovered inhibitor of inosine 5´-monophosphate
dehydrogenase (IMPDH) exhibits oncolytic activity.[2] IMPDH is the key enzyme of the de
novo guanylate biosynthesis including GTP and dGTP, and was shown to be linked with
proliferation. Therefore, IMPDH is a very good target for antitumor therapy. In order to be
active, benzamide riboside has to be converted to BAD, an NAD analogue that binds to the
NAD site on IMPDH. Inhibition of the enzyme by benzamide riboside selectively inhibits
tumor cells growth and induces apoptosis in various human tumor cell lines.
The discovery of tiazofurin (2-1) as a specific inhibitor of IMPDH with antitumor properties
led to a greater understanding of the IMPDH function and its importance in therapy.
O
OHHO
CONH2
HO
O
OHHO
HO
NS
CONH2
2-2
2-1
Scheme 2.1: Structure of tiazofurin (2-1) (TR) and benzamide riboside (2-2) (BR)
Benzamide riboside (2-2) (BR) was developed to find a better inhibitor of IMPDH.[1] BR
exhibited dual mechanisms of action; first by inhibiting IMPDH and second by inducing
apoptosis in highly proliferating cells, such as cancer. The previous investigation ensures that
benzamide riboside influences the adenosine-receptor-transport-activity. These unfavorable
influences should be reduced by chemical modifications. For a high biological selectivity in
the cells new derivatives must be synthesized, which have less structural similarity to NAD.
To prepare these molecules, a general method for the synthesis of benzamide riboside
derivatives must be developed.
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
8
2.2 Mechanism of Action of Benzamide Riboside
Benzamide riboside [(1-
β
-D-ribofuranosyl)benzene-3-carboxamide)] (2-2), a C-glycoside
analogue of nicotinamide riboside, has generated interest because of its excellent toxic
activity.[1] Benzamide riboside (BR) was examined for its activity against different human
tumor cell lines.[2] A detailed comparative study of the activity was conducted with
selenazofurin (SR) and tiazofurin (TR).[14] The mechanisms of action of SR and TR were
shown to be due to their conversion to an analogue of NAD that inhibits the NAD utilization
by IMPDH. This causes an arrest of cancer cell proliferation, leading to cancer cell
death.[15,16,17] The enzyme inosine 5´-monophosphate dehydrogenase (IMPDH) is responsible
for the formation of xanthine-monophosphate from IMP (inosine-monophosphate) and was
shown to be the rate limiting enzyme for the de novo formation of GTP (guanosine-
triphosphate) and dGTP (deoxyguanosine-triphosphate).[18] Due to the requirement of high
concentration of GTP and dGTP in rapidly proliferating cells, tumor cells have, in contrast to
slowly proliferating cells, high activity of IMPDH. First, Jackson showed in a spectrum of
hepatomas that the enzyme activity is increased linked with proliferation and malignant
transformation.[18] Therefore the enzyme is considered to be an excellent target for antitumor
therapy. Malignant cells can be killed without causing much harm to normal, slowly growing
cells.
2.3 Synthesis of Benzamide Riboside
2.3.1 Synthesis of C-glycosides
The study and synthesis of C-nucleosides has been extensively investigated owing to their
biological activity and potential as drug candidates for antiviral and anticancer therapy.[19,20]
Nucleosides are generally considered to be compounds which contain a heterocyclic aglycon
and a carbohydrate moiety that are joined together by a carbon–nitrogen bond. However, C-
nucleosides differ from the more common nucleosides in the way that the sugar and
heterocyclic aglycon are connected by a C–C, rather than a C–N bond. The C–C bond is
responsible for the resistance of C-nucleosides to hydrolytic and enzymatic cleavage.
Numerous synthetic strategies have also been investigated in order to optimize yields and
stereo selectivity in the glycosylation reaction. Methods for the synthesis of C-nucleosides
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
9
have been studied extensively;[21,22] however, synthetic obstacles in terms of low yield and/or
low stereoselectivity have been frequently encountered. Two major synthetic approaches to
C-nucleosides have been established:
(1) introduction of a functional group at the anomeric position of a sugar derivative, followed
by the construction of a heterocyclic base; and
(2) direct attachment of a pre-formed aglycon unit to an appropriate carbohydrate moiety.
The first synthetic approach, which has been reviewed in detail,[19,21] is a versatile method for
the preparation of C-nucleosides since numerous heterocyclic bases can be constructed
starting from an appropriate functional group at the anomeric carbon atom of a sugar
component. However, this approach involves a large number of steps, results in rather low
yields, and often results in unsatisfactory stereoselectivity. The possible methods for
preparation of C-glycosides are as follows:
¾ Coupling of ribofuranose derivatives with organometallic reagents
¾ Heck-type coupling reaction
¾ Coupling of protected ribofuranosyl chlorides with organometallic reagents
¾ Coupling of 1,4-ribonolactone derivatives with organometallic reagents
¾ Coupling reactions mediated by Lewis acids
2.3.2 Preparation of 2,3,5-tri-O-benzyl-γ-ribonolactone (2-7)
The preparation of benzamide riboside was performed as described in literature.[23] Starting
from D-ribose (2-3) lactone 2-7 could easily be prepared in a four step synthesis. In the first
step, the glycosidic OH of ribose 2-3 was methylated in methanol using H2SO4 as catalyst.[24]
Benzylation[25] of methyl glycoside 2-4 gave the protected ribose 2-5 in a yield of 89%. As
solvent abs. DMF was employed, NaH was used for deprotonation, benzyl bromide as
nucleophile and tetrabutyl-ammonium iodide as phase transfer catalyst (Scheme 2.2).
O
OHHO
HO
OMe
O
OBnBnO
BnO
OMe
O
OHHO
HO
OH CH
3
OH
H
+
NaH, BnBr
TBAI, DMF
2-3 2-4 2-5
Scheme 2.2: Transformation of D-ribose 2-3 to methyl-benzyl ether 2-5
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
10
Methyl glycoside protecting group of 2-5 was cleaved by HCl in dioxane to afford lactol 2-6,
which was used for the next step without purification. To prepare lactone 2-7, oxidation[26]
was performed in DMSO with acetic acid anhydride. After crystallization from ether/pentane,
lactone 2-7 was isolated as white needles in a yield of 85 %.
O
OBnBnO
BnO
OH
O
OBnBnO
BnO
O
HCl
dioxane
Ac
2
O
DMSO
2-6 2-7
Scheme 2.3: Preparation of lactone 2-7
The oxidation begins with the activation of DMSO through a nucleophilic attack of an oxygen
atom of the DMSO on one of the carbonyl carbon atoms of the acetic acid anhydride. This
step results in the formation of sulfoxonium ion A, which is the activated form of DMSO.
OO
O
S
O+
δ
δ
CH
3
COO
-
SOO
A
Scheme 2.4: Mechanism of the oxidation Part I.
In the next step, sulfoxonium ion A reacts with a hydroxyl group of lactol 2-6, that generates a
sulfurane B from which, after the cleavage of another acetate anion, sulfoxonium salt C is
formed.
OBnBnO
O
BnO
OHSOO
SOO
BnO
O
BnO
O
H
H
H
-H
+
SOO
O
BnO
O
HCH
3
COO
-
S
CH
2
OBn
BnO
O
BnO
O
H
H
CH
3
CH
3
COO
-
CH
3
COOH
OBn
BnO OBn
BC
2-6
Scheme 2.5: Mechanism of the oxidation Part II.
Acetate ion, which is now present in high concentration, serves as a base and deprotonates
one of the methyl groups of intermediate C. The obtained sulfonium-ylide D provides lactone
2-7 through
β
-elimination over a cyclic transition state.
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
11
S
CH2
OBn
OBn
O
OBn
O
CH3
H
OBn
OBn
O
OBn
O+(CH3)2S
D2-7
Scheme 2.6: Mechanism of the oxidation Part III.
2.3.3 Coupling of 1,4–Ribonolactone (2-7) with organometallic reagent 2-11
The key step of the synthesis is the coupling of the sugar moiety with the aromatic part and
the reduction of the tertiary hydroxyl group to achieve satisfactory
β/α
ratio with the
β
-
anomer 2-14 as the major product.
To protect the amide function of the target molecule 2-20, the oxazoline group was applied
for the coupling reaction with lactone 2-7, since it is stable against both Grignard- and
lithium-organic compounds. The preparation of oxazoline 2-10 was accomplished in a three
step synthesis starting from 3-bromo benzoic acid (2-8), which was transformed into acid
chloride 2-9 by treatment with SOCl2. The addition of 2-amino-2-methyl-propanol and ring
closure with SOCl2 afforded oxazoline 2-10.[27]
Br
COOH
Br
COCl
Br
O
N
SOCl
2
2-amino-2-methyl-propanol
CH
2
Cl
2
2-8 2-9
2-10
Scheme 2.7: Preparation of oxazoline 2-10
The lithiation of oxazoline 2-10 with butyllithium was carried out at −78 °C in abs. THF
under argon atmosphere. TLC monitoring of the reaction showed that the starting material
was completely consumed after 30 min. The following reaction at −78 °C between the
lithiated oxazoline 2-11 and the lactone 2-7 proceeded surprisingly fast, so that the reaction
mixture was worked up after 3 hours. This step gave cleanly the intermediate lactol 2-12,
which was not isolated, but directly subjected to the triethylsilane reduction. To assure the
complete conversion of the reduction, the reaction was left to warm up from −78 °C to +10 °C
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
12
overnight. After column chromatography only the sterically uniform
β
-C-riboside 2-14 was
isolated as the single product in 87 % yield over two steps.[28,29]
O
OBnBnO
BnO
O
N
Li
O
N
O
OBnBnO
BnO
O
2-7
OH
2-11
2-12
+
Scheme 2.8: Coupling reaction of the lithiated oxazoline 2-11 and lactone 2-7
During reduction of the hemiketal 2-12 with triethylsilane in the presence of Lewis acid
(BF3·OEt2), the nucleophile addition of the hydride ion occurred from the α- and accordingly
from the equatorial side of the oxonium ion 2-13. The stereochemical control can be
explained by the anomeric effect of the ring-oxygen and by the higher stability of the
β
-
anomer.
2-12 O
OBnBnO
BnO
O
N
O
OBnBnO
BnO
O
N
+
H
BF3 OEt2
.
Et3SiH
2-13 2-14
Scheme 2.9: Mechanism of the reduction by Et3SiH
2.3.4 Cleavage of the oxazoline group and transformation to benzamide riboside (2-2)
To prepare the amide function of the target molecule 2-2 the cleavage of the oxazoline group
was performed under basic conditions.[30] The desired C-ribofuranosylbenzoic acid 2-16 was
obtained by first heating oxazoline 2-14 with methyl iodide in nitromethane and then
refluxing the intermediate iodide salt 2-15 with 20 % methanolic KOH for 2 days.
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
13
2-14 O
OBnBnO
BnO
O
N
2-15
MeI
CH
3
NO
2
I
20% KOH
CH
3
OH
O
OBnBnO
BnO
COOH
2-16
Scheme 2.10: Cleavage of the oxazoline protecting group
Acid 2-16 could easily be transformed to the acid chloride 2-17 by heating it with SOCl2
without any solvent. According to the traditional Schotten-Baumann reaction,[31] treatment of
acid chloride 2-17 with ammonia resulted in amide 2-18, which readily crystallized from
ether/pentane after column chromatography.
O
OBnBnO
BnO
COCl
2-16
2-17
SOCl
2
O
OBnBnO
BnO
CONH
2
2-18
NH
3
Scheme 2.11: Transformation of acid 2-16 to amide 2-18
The cleavage of the benzyl ether was performed by hydrogenolysis with palladium/charcoal
as catalyst at room temperature under normal pressure. Changing the polarity of the solvent
had a very strong influence on the product composition. If hydrogenation was carried out in
THF, without any additional methanol, then either no product was formed, or the reaction
took place very slowly. By adding a large amount of methanol to the reaction mixture the
benzyl amide 2-18 was converted into the open chain benzamide riboside 2-19 exclusively. If
only a few drops of methanol were added to the THF solution, the reaction was complete in
3−4 hours. Just 5−10 % open chain side product was produced, and the desired target
molecule 2-2 was obtained in a good yield.
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
14
O
OBnBnO
BnO
CONH
2
2-18
O
OHHO
HO
CONH
2
2-2
OH
OHHO
HO
CONH
2
2-19
H
2
/ Pd (C) H
2
/ Pd (C)
THF,
few CH
3
OH
THF,
much CH
3
OH
Scheme 2.12: Hydrogenation of benzyl ether 2-18
The separation of the open-chained 2-19 and main product 2-2 was performed by column
chromatography on silica gel with gradually increasing the polarity of the eluting solvents.
The products were identified by NMR analysis.
2.4 Synthesis of Benzamide Riboside Analogues
2.4.1 Preparation of 3-deoxy sugar derivative (2-32)
Since the key step of the synthesis is the coupling of a sugar-lactone with oxazoline 2-10, the
preparation of the 3-deoxy-ribonolactone (2-26) was necessary in the first step.
Commercially available xylose (2-20) was used as starting material, because it is much
cheaper than the C-3-epimeric ribose. During the synthesis the C-3 hydroxyl group of xylose
was removed by the DeLederkremer method[32] in order to obtain the desired 3-deoxy sugar
derivative.
2.4.1.1 Synthesis of 3-deoxy-ribonolactone (2-26)
Selective oxidation of the anomeric hydroxyl group was carried out in aqueous solution with
bromine[33] to afford the lactone 2-21. After completion of the reaction (5 days), the excess of
bromine was extracted with diethyl ether into the organic phase, and the water phase
containing the lactone 2-21 was lyophilized. Treatment of lactone 2-21 with benzoyl chloride
in pyridine/chloroform afforded benzoyl ester 2-22 in good yield.
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
15
O
OH
OH O
OH
HO
O
Br2
H2O
BzlCl
pyridine
O
OBzl
BzlO
O
2-20 2-21 2-22
HO
OH OH OBzl
Scheme 2.13: Conversion of xylose 2-20 to benzoyl ester 2-22
The reduction of the esterified butyrolactone 2-22 in basic medium with Raney nickel or
palladium-charcoal[34] gave rise to
β
-elimination and the intermediate enol lactone 2-23 was
subsequently reduced by hydrogenation of the endocyclic bond to deoxy lactone 2-24.
2-22
O
OBzl
BzlO
O
2-23
O
OBzl
BzlO
O
2-24
H
2
, 10% Pd/C
Et
3
N, EtOAc
O
OBzl
O
BzlO
OBzl
Scheme 2.14:
β
-elimination followed by hydrogenation of ester 2-22
Deoxygenation was accomplished in a yield of 68 % over two steps; and product 2-24
crystallized readily from ethanol.
However, the ester function reacts with metalloorganic reagents. Therefore, lactone 2-24 was
converted to benzyl ether 2-26. To cleave the benzoyl ester group, Zemplén saponification
was applied using freshly prepared sodium methanolate in methanol to obtain unprotected 3-
deoxy-ribonolactone 2-25. TLC monitoring showed that the reaction was completed in 30
minutes. The reaction mixture was worked up by addition of acidic ion exchange resin to the
solution, and after filtration the solvent was evaporated. Without further purification,
benzylation of 2-25 was performed in abs. DMF using NaH for deprotonation of the alcohol
function, benzyl bromide as nucleophile, and TBAI as phase transfer catalyst. Benzyl ether 2-
26 could be isolated as an oil (yield 46 %).
O
OH
HO O
2-25
O
OBn
BnO
O
2-26
2-24 NaOMe
MeOH
NaH, BnBr
TBAI, abs. DMF
Scheme 2.15: Zemplén reaction of 2-24 and benzylation to ether 2-26
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
16
2.4.1.2 Coupling of 1,4–Ribonolactone 2-26 with organometallic reagent (2-11)
Using the conditions of the previously mentioned metalloorganic reaction,[29,30] the coupling
between bromo oxazoline 2-10 and 3-deoxy lactone 2-26 was performed in abs. THF at
−78 °C. Lithium oxazoline 2-11, generated from oxazoline 2-10 by treatment with n-
butyllithium, was reacted in situ with 3-deoxy lactone 2-26 to afford the tertiary alcohol 2-27
with a yield of 67 %.
Li
O
N
O
OBn
O
2-26
2-11 2-27
+
BnO O
OBn
BnO
OH
O
N
Scheme 2.15: Coupling reaction of lithiated oxazoline 2-11 and lactone 2-26
Deprotonation and removal of the tertiary OH group took place under the same reaction
conditions as described for BR. Due to thermodynamic control the reduction could be
performed stereoselectively.
2-27 BF3 OEt2
.
Et3SiH
2-28 2-29
O
OBn
BnO
O
N
+
H
O
OBn
BnO
O
N
Scheme: 2.16: Mechanism of reduction by Et3SiH
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
17
2.4.1.3 Transformation of C-glycoside 2-29 to 3-deoxy benzamide riboside (2-32)
Basic cleavage of the oxazoline protecting group resulted in acid 2-30, which was purified by
column chromatography on silica gel and then treated with thionyl chloride and subsequently
with aqueous ammonia solution to obtain amide 2-31 in a yield of 88 % over 2 steps.[31]
2-29 1) MeI
2) KOH
1) SOCl
2
2) NH
3
2-30 2-31
O
OBn
BnO
COOH
O
OBn
BnO
CONH
2
Scheme 2.17: Preparation of benzyl protected 3-deoxy ribose phenyl amide 2-31
Reductive cleavage of the benzyl ether by careful hydrogenation of 2-31 was carried out in
abs. THF containing 5 % of ethanol. The reaction proceeded fast; TLC monitoring showed the
complete consumption of the starting material 2-31 in 2 hours.
H
2
, Pd / C
2-31
2-32
THF, EtOH
O
OH
HO
CONH
2
Scheme 2.18: Reductive cleavage of benzyl ether 2-31
3-Deoxy-benzamide riboside (2-32) was isolated in a yield of 86 %. The formation of open-
chain side product was not observed.
2.4.2 Preparation of derivatives with different substituents on the aromatic ring
New C-glycosides were prepared by coupling of lactone 2-7 with different meta-substituted
bromo benzene derivatives in order to investigate if the amide function of the benzamide
riboside (2-2) is necessary for the antitumor activity.
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
18
2.4.2.1 Synthesis of
β
-D-ribofuranosyl-benzene (2-35)
Primarily unsubstituted bromo benzene (2-33) was used in the same procedure[29,30] as
mentioned before (see Chapter 2.2.3). The coupling reaction was performed in abs. THF at
−78 °C and proceeded quickly. The reduction took place in dry dichloromethane by the
addition of triethylsilane and boron trifluoride diethyl etherate giving an over-all yield of
67 % for the two steps.
O
OBnBnO
BnO
O
OBnBnO
BnO
2-7
O
Br
+1) BuLi, abs THF
2)Et
3
SiH, BF
3
OEt
2
.
2-33 2-34
Scheme 2.19.: Preparation of C-glycoside 2-34
Hydrogenation of benzyl ether 2-34 in abs. THF / 5 % methanol unfortunately did not lead to
the desired ribofuranyl benzene (2-35). Only partially hydrogenated products were observed.
Even increasing the polarity of the solvent did not result in the target molecule 2-35. The
cleavage of the benzyl ether protecting groups was therefore carried out by acidic cleavage
using BBr3 as a Lewis acid.
O
OHHO
HO
2-35
2-34 BBr
3
abs. CH
2
Cl
2
Scheme 2.20: Acidic cleavage of benzyl ether protecting group
Starting material 2-34 was entirely consumed after the reaction time of 10 min. at 0 °C. The
reaction mixture was then neutralized by the addition of saturated aqueous NaHCO3 solution
and the water phase was evaporated under reduced pressure. The resulting white crystals
(product 2-35 and inorganic salts) were dissolved in methanol and filtered. Ribofuranyl
benzene (2-35) was isolated as the only product with a yield of 92 %.
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
19
2.4.2.2 Synthesis of
β
-D-ribofuranosyl-3´-fluorobenzene (2-38)
Since several fluoro sugar derivatives are potential anticancer and anti human immuno-
deficiency virus agents (HIV),[35,36] the preparation of a fluoro derivative of benzamide
riboside (2-2) was worth trying. Coupling reaction of commercially available 1-bromo-3-
fluorobenzene (2-36) with ribonolactone 2-7 afforded the C-glycoside 2-37 with a good yield
(83 %, after reduction of the tertiary hydroxyl group by BF3·OEt2, Et3SiH). Because aryl
bromide reacts considerably faster with n-BuLi than aryl fluoride no other product was
isolated from the reaction mixture.
O
OBnBnO
BnO
O
OBnBnO
BnO
2-7
O
Br
+1) BuLi, abs THF
2)Et
3
SiH, BF
3
OEt
2
.
2-36 2-37
F
F
Scheme 2.21: Coupling reaction of 1-bromo-3-fluorobenzene (2-36) with ribonolactone 2-7
As the last step, the cleavage of benzyl ether was performed. Also in this case, hydrogenation
was not successful. However, using BBr3 as Lewis acid in dry dichloromethane, the target
compound 2-38 could be obtained after purification with a yield of 75 %.
O
OHHO
HO
2-38
2-37 BBr3
abs. CH2Cl2
F
Scheme 2.22.: Preparation of
β
-D-ribofuranosyl-3´-fluorobenzene (2-38)
2.4.2.3 Synthesis of
β
-D-ribofuranosyl-m-anisol (2-41)
For use in the following investigation on structure activity relationship of benzamide riboside
(2-2), a strong electron donating substituent in meta position was applied. 3-Bromoanisol (2-
39) was coupled with ribonolactone 2-7 and the intermediate tertiary alcohol was reduced.
(see Chapter 2.2.3).
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
20
O
OBnBnO
BnO
O
OBnBnO
BnO
2-7
O
Br
+1) BuLi, abs THF
2)Et
3
SiH, BF
3
OEt
2
.
2-39 2-40
OCH
3
OCH
3
Scheme 2.23.: Coupling of 3-bromoanisol (2-39) and ribonolactone 2-7
Due to the fact that the activation energy of the cleavage of a benzyl group by a Lewis acid is
lower then that of a methyl ether, the selective debenzylation could be performed at −78 °C
using BBr3. TLC monitoring showed that the reaction was completed after 1.5 hours at this
temperature. After neutralization by the addition of aqueous NaHCO3 solution, the target
compound 2-41 could be isolated from the water phase. Using a small column of silica gel for
purification,
β
-D-ribofuranosyl-m-anisol was obtained in 85 % yield.
O
OHHO
HO
2-41
2-40 BBr3
abs. CH2Cl2
OCH3
Scheme 2.24.: Debenzylation to 2-41
2.5 Investigation Towards the Synthesis of 2-deoxy- and 2,3-dideoxy-
Benzamide Riboside
2.5.1 Preparation of 2,3-dideoxy-5-benzyl-ribonolactone (2-46)
2,3-Dideoxy ribonolactone (2-45) was prepared from L-glutamic acid (2-42) as described in
the literature.[37] In the presence of HCl, glutamic acid (2-42) was treated with aqueous
NaNO2 solution; HNO2 was generated during this process. Protonation and cleavage of a
water molecule resulted in a mesomerically stabilized nitrosyl cation. In the next step a
nucleophile attack of the amine on the nitrosyl cation intermediate took place and a
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
21
nitrosoamine salt was formed. After several deprotonation and protonation steps, H2O was
eliminated and a diazonium ion is formed.
HOOC
HOOC
N
H
H
NO NO
N
HOOC
HOOC
H
H
+N
HOOC
HOOC
N
2-42
Scheme 2.25: Formation of diazonium ion
Nitrogen is eliminated to give a very reactive cation. Ring closure occured and subsequent
deprotonation yielded acid 2-43.
- N2O
O
H
HOOC
H
OO
H
HOOC
H
OO
H
HOOC
-H
2-43
Scheme 2.26: Ring closure to acid 2-43
The acid 2-43 was reduced to the corresponding alcohol in two steps. Esterification was
performed in benzene/ethanol using p-toluene sulfonic acid as catalyst and resulted in the
formation of 2-44a and open chained diester 2-44b.
HO
H
COOEt
EtOOC
+
2-43
OO
EtOOC
2-44a 2-44b
Scheme: 2-27: Esterification to 2-44a and 2-44b
The separation of cyclic- 2-44a and open-chained esters 2-44b was not necessary because
during the reduction with sodium borohydride in ethanol the open-chained product 2-44b
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
22
cyclisized to 2-44a and secondary alcohol 2-45 was isolated as a single product of the
reduction.
OO
O
EtO
O
H
Et
OO
O
EtO
H
α
β
γδ+
+ EtO
-
2-44b
OO
EtOOC
2-44a
Scheme: 2-28: Ring closure of ester 2-44b to 2-44a
The benzylation of the secondary alcohol 2-45 was performed in abs. DMF using freshly
prepared Ag2O as a base instead of NaH to avoid the opening of the lactone ring.[38]
NaBH
4
OO
2-45
HO
Ag
2
O, BnBr
abs. DMF
OO
2-46
BnO
Scheme 2.29: Preparation of 2,3-didesoxy-5-benzyl-ribonolactone (2-46)
2.5.2 Preparation of 2-deoxy-3,5-dibenzyl-ribonolactone (2-52)
Commercially available 2-deoxy ribose (2-47) was employed as starting material. Using the
previously mentioned oxidation procedure,[33] 2-deoxy (2-47) ribose was converted into 2-
deoxy ribonolactone (2-48) (see Chapter 2.3.1.1).
O
HO
HO
OH O
HO
HO
O
Br
2
2-47 2-48
H
2
O
Scheme 2.30: Oxidation to 2-deoxy-lactone 2-48
For benzylation, the trichloroacetimidate procedure of Schmidt was chosen.[39] The
benzylation agent benzyl-2,2,2-trichloroacetimidate (2-51) was prepared from benzyl alcohol
(2-49) and trichloroacetamide (2-50) in dry diethyl ether under acidic conditions. The imide
formation proceeded smoothly within 10 minutes.[40]
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
23
O
NH
Cl
Cl
Cl
CH
2
OH
+H
2
N
Cl
Cl
Cl
O
2-49 2-50 2-51
Scheme 2.31: Preparation of benzyl-2,2,2-trichloroacetimidate (2-51)
2-Deoxy-3,5-dibenzyl-ribonolactone (2-52) was obtained after purification in a very good
yield by treating diol (2-48) with benzyl-2,2,2-trichloroacetimidate (2-51) as benzylation
agent in the presence of trifluoromethanesulfonic acetic at room temperature.
Trichloroacetamide (2-53) that formed during the reaction crystallized readily and could be
separated by filtration.
O
HO
HO
O
2-48
O
BnO
BnO
O
2-52
2-51, TFA
abs. dioxane +H
2
N
O
Cl
Cl
Cl
2-53
Scheme 2.32: Benzylation of ribonolactone 2-48 by benzyl-2,2,2-trichloracetimidate (2-51)
2.5.3 Investigation on the coupling of deoxy-lactones 2-46 and 2-52 with lithiated
oxazoline 2-11
Both ribonolactone derivatives 2-46 and 2-52 were subjected to metalloorganic coupling
reaction with lithiated oxazoline 2-11 in order to prepare the C-glycosides 2-54 and 2-55
using the methods mentioned in Chapter 2.2.4.
Li
O
N
2-11
O
BnO
BnO
O
N
O
BnO
O
N
2-54
2-55
2-522-46
Scheme 2.33: The attempted coupling of metal-organic reagent 2-11 with the lactones 2-46 and 2-52
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
24
Unfortunately no transformation was observed. In the literature the coupling reaction of
various substituted lithiated aryl compounds with cyclic or open chained ketons was
performed by the addition of TMEDA[41,42]. However, the addition of TMEDA to the reaction
mixture in the case of both metalloorganic coupling reactions did not yield the desired
products 2-54 and 2-55.
This reaction of lithiated aryls with lactones was a convenient method for the preparation of
benzamide riboside (2-2), 3-deoxy-benzamide riboside (2-32), and BR-derivatives 2-35, 2-38,
2-41, but gave no product for 2-46 and 2-52. The failure could be explained by the absence of
a benzyloxy substituent in position C-2 of ribonolactone. Evidently, this group plays a
significant role during the reaction. The existence of the neighboring group effect could
provide an explanation. Probably the oxygen atom of the benzyloxy group participated in the
coupling reaction in a way (chelat formation), that the presence of it is indispensable to
perform the reaction.
2.6 Biological test results
The cytotoxicity of compounds 2-2, 2-32, 2-35, 2-38, 2-41 were examined against both
human myelogenous leukemia K562 cells and human colon carcinoma HT-29 cells.
Benzamide riboside (2-2), at a concentration which kills about 20 % of the lymphoma cells,
only slightly increases the toxicity of dexamethasone on 549.1 lymphoma cells (in contrast to
benzamide). Therefore, it appears that the poly(ADP-ribose) synthetase is not preferentially
affected by benzamide riboside (2-2) or the benzamide containing NAD analogue compared
to other NAD-dependent enzymes. Although benzamide riboside (2-2) does not potentiate the
toxicity of glucocorticoids in S49.1 lymphoma cells, it could be an interesting agent in
antitumor cell therapy. Since the concentration of pyridine dinucleotides in malignant cells
has been shown to be low as compared to normal tissue, tumors could be more susceptible to
compounds such as benzamide riboside (2-2). The riboside (2-2) is presently being tested for
antitumor activity in vivo. The compound did not show any stimulatory or suppressive
behavior in immunomodulation tests with human lymphocytes.
It was shown that 5 µM BR (2-2) induces apoptosis of HL-60 cells. Increasing the
concentration to 20 µM BR provokes necrosis of HL-60 cells, which is reflected by typical
membrane and organelle disintegration. A rapid and sustained decrease in the intracellular
concentration of GTP and dGTP was observed. 10µM BR (2-2) treatment of K562 cells for 2
Synthesis of Benzamide Riboside Derivatives with Antitumor Activity
25
hours significantly decreases GMP, GDP, GTP and dGTP levels without changing the ATP
level.
3-Deoxy benzamide riboside (2-32), and derivatives 2-35, 2-38, 2-41 did not show inhibition
both in human myelogenous leukemia K562 and human colon carcinoma HT-29 cells up to
100 µM concentration. That is, both the amide function of the benzene ring and the C-2
hydroxyl group are necessary for the antitumor activity.
Benzamide riboside (2-2) exhibits potent antitumor activity against a diverse panel of human
cancer cells. The cytotoxic potency is time related and dependent on duration of exposure to
the drug. Benzamide riboside (2-2) might be a new addition as a promising antitumor agent.
Further in vivo studies in animals should provide the basis for future clinical studies.
Synthesis of Benzamide Riboside Adenine Dinucleotide
26
3 Synthesis of Benzamide Riboside Adenine Dinucleotide
This chapter focuses on the chemical synthesis of benzamide riboside adenine dinucleotide
(BAD) (3-5). The enzymatic synthesis from benzamide riboside 5´-monophosphate (3-3) and
ATP (adenosine triphosphate) has already been described in the literature,[43] utilizing a
partially purified commercially available preparation of NAD pyrophosphorylase from hog
liver. In cooperation with the Max Plank Institute for Biophysics in Frankfurt, Germany
enzyme-substrate complex studies will be performed using BAD (3-5), to investigate the
active site of various reductase enzymes.
3.1 Introduction
Benzamide riboside (2-2) is related to NAD (3-1) (nicotinamide adenine dinucleotide), in
which the pyridine ring of NAD is replaced by a benzene ring. However, benzamide riboside
(2-2) binds to the NAD bonding site of enzymes. NAD plays a very important role in many
biochemical processes. NADH formed during the glycolysis is reoxidised under anaerobic
conditions. In the citric acid cycle, it acts as a coenzym of the isocitrate-dehydrogenase, which
catalyses the reduction to α-ketoglutarate. Since benzamide riboside is a C-glucoside, it is
unable either to accept or to donate protons. This way the biological processes are stopped
and the cell dies.
O
CONH
2
HO OH
HO
ON
CONH
2
HO OH
OPO
O
OH
P
OH
O
O
O
N
N
N
NH
2
N
OHHO
2-2 3-1
Scheme 3.1: Structure of benzamide riboside (2-2) and NAD (3-1)
In order to accomplish these studies, the NAD analogue BAD (benzamide riboside adenine
dinucleotide) (3-5) was prepared in vitro.
Synthesis of Benzamide Riboside Adenine Dinucleotide
27
3.2 Synthesis of benzamide riboside adenine dinucleotide (3-5)
The starting material of this synthesis was the target compound of the previous Chapter 2.3.
To get monophosphate 3-3, the tertiary OH-groups of benzamide riboside (2-2) were
protected by an isopropylidene group, which was prepared in acetone and a few drops of
H2SO4 was applied as a catalyst.
O
CONH
2
HO OH
2-
O
3
PO
3-3
O
CONH
2
O O
HO
2-2 H
2
SO
4
aceton
POCl
3
(EtO)
3
P
3-2
Scheme 3.2: Preparation of monophosphate 3-3
Phosphorylation of 2-2 and cleavage of the acetonide group proceeded in a one-pot reaction
after the standard method of Ludwig[44] to generate the monophosphate 3-3. The coupling of
the monophosphate 3-3 with adenosine monophosphate (3-4) was carried out by Michael
Morr in Braunschweig at the GBF, Germany according to the method of Marquez et al.[45,46].
This procedure consisted of activating one of the nucleotides (as usual, the five-membered
base nucleotide) with carbonyldiimidazole.
The formation of the imidazolidates was easily monitored by HPLC and required no isolation.
When the reaction was completed, methanol was added to hydrolyze the excess of reagent,
followed immediately by the addition of AMP (3-4) as a DMF solution containing tri-n-
butylamine. Formation of the dinucleotide was complete after 48 h. The aqueous extract
containing both the product 3-5 and its 2',3'-cyclic carbonate analogue, was treated by
triethylamine to convert it to the deblocked product 3-5.[47]
In those cases where this last step was omitted, significant amounts of the corresponding
cyclic carbonate dinucleotide was obtained as verified by MS analysis. After the triethylamine
treatment product 3-5 was purified by chromatography on Hamilton X4-H4 anion exchange
column.
Synthesis of Benzamide Riboside Adenine Dinucleotide
28
O
CONH
2
HO OH
OPO
O
OH
P
OH
O
O
O
N
N
N
NH
2
N
OHHO
P
OH
O
O
ON
N
N
NH
2
N
HO OH
HO 3-3
3-4 3-5
Scheme 3.3: Preparation of BAD (3-5)
3.3 Enzym-substrate complex studies
Due to the high similarity between BAD (3-5) and NAD (3-1), BAD (3-5) also binds to the
active site of NAD binding enzymes. However, BAD (3-5) probably binds irreversible, since
it can neither accept nor donate protons. For this reason 3-5 is a suitable substrate for enzyme-
substrate complex studies. X-ray crystallographic studies of the complex with reductase will
be performed by Dr. Carola Hunte at the Max Plank Institute for Biophysics in Frankfurt,
Germany. The combined approach of X-ray crystallography, biochemical analysis, site-
directed mutagenesis and spectroscopy will provide valuable information about the active site
of the enzyme. If the active site of the reductase is known, further benzamide riboside
derivatives, with a better selectivity in tumor cells, can be developed. On the other hand the
molecular mechanism of action of the reductase can be explored, since there are several
possible pathways for proton uptake and release.
Synthesis and Structure-Activity Relationship of Antifungal Coniothyriomycin Analogues
29
4 Synthesis and Structure-Activity Relationship of Antifungal
Coniothyriomycin Analogues
4.1 Introduction
In the research group of Prof. Krohn, an open-chain imide named coniothyriomycin (4-1) with
remarkable antifungal activity was isolated from an unidentified fungus Coniothyrium sp.[48,49]
NOCH3
OH
Cl O O
OH
coniotyriomycin (4-1)
Figure 4-1.: Structure of coniothyriomycin (4-1)
Unfortunately, these open-chain mixed amides of phenylacetic and fumaric acid, in spite of
excellent short-term antifungal activity, did not show curative effects. One reason for this was
the inherent instability of the imide functionality in the presence of nucleophiles such as
water. Therefore, in the hope to increase the chemical stability and retain or even increase
antifungal activity, different coniothyriomycin derivatives were prepared to examine the
structure-activity relationship. This way, we extended our study to the preparation and
biological testing of various analogues by systematically changing the structure of 4-1 by:
¾ replacement of the substituted phenylacetic acids with substituted benzoic acids,
¾ changing of hydrophobicity by variation of the alcohol component,
¾ variation in the degree of saturation of the fumaric acid moiety,
¾ replacement of carbon by nitrogen, oxygen or sulfur in the middle part of the
molecule,
¾ incorporation of the open-chain part of the molecule into cyclic arrangements,
¾ changing the substitution pattern of the aromatic ring.
Synthesis and Structure-Activity Relationship of Antifungal Coniothyriomycin Analogues
30
4.2 Developing a new method for the synthesis of mixed imides
In the previous syntheses of coniothyriomycin analogues[48] the method of Suhara et al.[50]
was used, starting from nitriles via the corresponding ethyl imidates. However, the yields
using this method were poor (10–36 %) and the workup of the dark brown reaction mixture
was tedious.
The efforts were therefore directed towards a simplification of the procedure. To that end,
equivalent amounts of benzamide (4-2) were heated with the fumaric monoethyl ester
chloride[51] (4-3a) in toluene (Scheme 1). However, starting with a 1:1 ratio, only modest
yields of the desired mixed imide 4-5 were observed and the mixed anhydride 4-4 was
identified as the major side product.
Evidently, both theoretically possible reaction pathways a and b were realized: attack by the
amide nitrogen on the acyl chloride (path a) to form 4-5a, and attack by the nucleophilic
oxygen (path b) to form 4-4. However, the acyl group transfer potential was still preserved in
the mixed anhydride 4-4 and attack by excess benzoic acid amide {benzamide} (path c) on
the mixed anhydride carbonyl might ultimately result in the formation of the mixed open
chain imide 4-5a.
In fact, using two equivalents of amide 4-2, pathway c was followed consuming the mixed
anhydride 4-4, and resulted in the mixed imide 4-5a. Yields of 50 % and more were achieved
in a clean, easily worked-up reaction.
Interestingly, the formation of the symmetric benzoic acid imide, resulting by attack of the
nitrogen on the benzoic acid part of anhydride 4-4, was not observed.
NH2
O
+
Cl OEt
O
ONOEt
O O
OH
path c
OOEt
O O
O
mixed anhydride 4-4
path a
path b
4-3
4-5a
4-2 path a
path c
Scheme 4-1: Formation of mixed imide 4-5a starting from amide 4-2 and acid chloride 4-3a via the
mixed anhydride 4-4
Synthesis and Structure-Activity Relationship of Antifungal Coniothyriomycin Analogues
31
The newly developed method proved to be general and most of the mixed imides (4-5a to 4-
5h, 4-10 to 4-14, 4-21), were prepared using this simple procedure. However, in some cases it
was advantageous to use the anion of amides such as 4-2. Also, the yields were generally
higher starting with benzamide (4-2) than with phenylacetic acid amide (4-6a) (Scheme 4-2).
4.3 Synthesis of coniothyriomycin analogues – Variation of the
substitution pattern of the benzyl-ring
The first series of compounds were a number of mixed benzoic acid and fumaric ester open
chain imides with variation in the substituents in the aromatic ring and the ester alcohol
component. It was investigated whether omission of the methylene group in 4-5a by
replacement of phenylacetic acid in the coniothriomycin analogues[48] with benzoic acid
would preserve their antifungal activity. In addition, variation of the substituents from
methoxy to fluoride or nitro groups in 4-5b to 4-5g (Figure 4-2) would show the influence of
electron density on biological activity. Furthermore, the lipophilicity of the fungicide was
increased in 4-5h by linking the n-octanyl ester of fumaric acid chloride to benzamide. The
data and substituents of the mixed benzoic acid-fumaric ester imides 4-5a to 4-5h are listed in
Table 4-1.
4-5a to 4-5h
NOR5
O O
OH
R1
R2
R3
R4
1
2
3
5
4
6
1' 2'
3' 4' 5'
Figure 4-2: Mixed benzoic acid-fumaric ester imides 4-5a to 4-5h
R
1 R
2 R
3 R
4 R
5 mp (°C) yield (%)
4–5a H H H H C2H5 87–89 °C 51 %
4–5b F H H H C2H5 54-55°C 42 %
4–5c H H NO2 H C2H5 135-137°C 44 %
Synthesis and Structure-Activity Relationship of Antifungal Coniothyriomycin Analogues
32
4–5d OCH3 H H H C2H5 98-100°C 46 %
4–5e H H OCH3 H C2H5 120-122°C 45%
4–5f H OCH3 H OCH3 C
2H5 162-164°C 43 %
4–5g H OCH3 OCH3 OCH3 C
2H5 114-116°C 65%
4–5h H H H H n-C8H15 oil 33 %
Table 4-1: Data and substituents of mixed benzoic acid-fumaric ester imides 4-5a to 4-5h
4.4 Synthesis of coniothyriomycin analogues – Changing the degree of
saturation, and hydrophopbicity in the fumaric ester side-chain
Next, the variation in the degree of saturation in the fumaric ester part was analyzed, starting
from phenylacetic amide as well as from benzoic acid amide. Omission of a methine group,
e.g. the shift from fumaric ester to malonic ester, or replacement with benzoic acid
(incorporating the double bond of fumaric acid into a ring system) was also in line with this
type of variation and worth testing for fungicidal activity. The construction of the mixed
imides 4-10 to 4-14 by coupling of amides 4-2 and 4-6a,b with the acid chlorides 4-7 to 4-9 is
shown in Scheme 4.2, employing about two equivalents of the respective amides as outlined
in Scheme 4.1. The phenolic imide 4-11c, as present in the natural product coniothyriomycin
(4-1), was prepared by reaction of benzyl ether 4-6b with 4-7, followed by hydrogenolysis of
benzyl ether 4-11b to 4-11c in order to evaluate the influence of a phenolic hydroxy group on
activity.
Synthesis and Structure-Activity Relationship of Antifungal Coniothyriomycin Analogues
33
NH2
OCH3
O
O
Cl
O
OCH3
O
Cl
O
NH2
O
or
or
or
4-2
4-6a,b
4-7
O
Cl
4-8
4-9
NOCH3
O O
OH
NOCH3
O O
OH
N
O O
H
O
OCH3
+
N
O O
H
4-10
4-11a,b,c
4-12
4-13: R = 3-OCH34-14: R = 4-OCH3
R
1
2
3
4
toluene
reflux
R
a: R = H, b: R = OBn, c: R = OH
R
Scheme 4-2: Construction of mixed imides 4-10 to 4-13 by coupling of amides 4-2 and 4-6a,b with
the acid chlorides 4-7 to 4-9.
4.5 Synthesis of coniothyriomycin analogues - Replacement of carbon by
nitrogen, oxygen or sulfur in the middle part of the molecule
Next, we investigated the replacement of carbon by nitrogen, oxygen or sulfur in the middle
part of the molecule to study the effect of these exchanges on bioactivity. Three different
types of compounds resulted from these exchanges: The bis-acylated hydrazine 4-16, the
acylated hydroxamide 4-18, and the acylated phenylhydrazines 4-20a to 4-20c. The
compounds were prepared by reaction of the hydrazide anion of 4–15, the hydroxamic acid 4-
4-17 or the phenylhydrazines 4-19a,b with the acid chlorides 4-3 or 4-3c, respectively, as
outlined in Scheme 4-3.
Synthesis and Structure-Activity Relationship of Antifungal Coniothyriomycin Analogues
34
N
H
OEt
O
O
O
N
N
H
N
H
O
O
OR'
N
H
OEt
O
O
O
O
H
N
H
O
NH21: KOH
2. + 4-3a
in CH2Cl2
N
H
O
OH
toluene
+ 4-3a
4-15 4-16
4-17 4-18
NH2
N
H
4-19a,b 4-20a-c
+ 4-3a CH2Cl2
30 min
R
a: R = H, R' = Et
b: R = 2,4-di-NO2, R' = Et
c: R = H, R' = n-C8H15
R
or
+ 4-3c
Cl OR'
O
O
4-3a,c
r. t.
Scheme 4-3: Synthesis of bis-hydrazide 4-16, acyl hydroxamic acid 4-18 and fumaric hydrazides 4-
20a-c
4.6 Biological Studies
The tested synthetic coniothyriomycin analogues showed primarily control of plant diseases
caused by representative fungi belonging to the class of Oomycetes, for example late blight on
tomatoes caused by Phytophthora infestans or downy mildew on grape vine caused by
Plasmopara viticola. The fungicidal activity was tested in vitro in 96-well microtitre plates
and on intact plants in a greenhouse.
The change of the molecular fragment phenylacetic amide to benzoic amide in 4-5a to 4-5h
retained the same good in vitro activity as the lead structure coniothyriomycin. 4-5a to 4-5d
controlled P. infestans with ED90-values of less than 0.5 ppm, 4-5e even at 0.125 ppm. Some
additional control of the casual organism of rice blast, Pyricularia oryzae, and of the casual
organism of leaf blotch on wheat, Septoria tritici was observed for 4-5a to 4-5e with ED90-
values of less than 2 and 8 ppm respectively. The fungicidal in vitro activity could only
partially be translated into in vivo activity in greenhouse tests on intact plants. Only 4-5f
Synthesis and Structure-Activity Relationship of Antifungal Coniothyriomycin Analogues
35
showed some initial protective control of P. infestans on tomatoes with additional initial good
protective activity against P. viticola on grapes. The compounds 4-5d, 4-5g and 4-5h showed
only some moderate control of P. viticola.
-20
0
20
40
60
80
100
120
140
12531820,50,1250,031
ppm
% growth
Phy tin
Botrci
Py r ior
Septtr
Scheme 4-4: Biological activity of 4-5a.
The hydrogenation of the double bond in the fumaric acid fragment, which resulted in 4-10
and 4-11a, led to loss of fungicidal activity both in vitro and in vivo. The other structural
variations in the compounds 4-13 and 4-14 resulted in reduced in vitro fungicidal activity
against P. infestans, with ED90-values of 31 and 8 ppm respectively. All other structural
variations in compounds 4-12, 4-16, 4-18, 4-20a to 4-20c led to a loss of any significant in
vitro or in vivo fungicidal activity.
Structure-Activity Relationships in Allergic Contact Dermatitis
36
5 Structure-Activity Relationships in Allergic Contact
Dermatitis
5.1 Introduction
Non-terpenoid and diterpenoid phenanthrenequinones (PAC) have been found in the plant
kingdom. Some of them occur in plants used in traditional Chinese medicine like Tan-Shen,
while others are constituents of orchids that are popular as ornamental plants. Quinones are
found mainly in plants, fungi, bacteria and the animal kingdom. By far the largest group in
plants involves the anthraquinones, benzoquinones and naphthoquinones.
Phenanthrenequinones (PAC) of non-terpenoid origin are rarely detected, and some of them
are simply oxidation products from their hydroxylated phenanthrene precursors. Currently
known naturally occurring 1,4-phenanthrenequinones have been isolated from species of the
orchid family, (denbinobin from Dendrobium nobile Lindl. and other orchid species,[52,53,54]
cypripedin from Cypripedium calceolus L.),[55] Annonaceae (annoquinone-A from Annona
montana MacFad.),[56] Labiatae (plectranthrones from Plectranthus species)[57,58] and
Combretaceae (combretastatin C-1 from Combretum sp.).[59] Also tropical timbers of the
Leguminosae-Papilionaceae family contain phenanthrenequinones such as latinone (in
Dalbergia latifolia Roxb.)[60] and melatinone (in D. melanoxylon Guill. & Perr).[61]
A greater number of structurally related diterpenoid and abietanoid quinones, like the
tanshinones, royleanones and miltirones, do occur in species of the Dioscoraceae, Lamiaceae,
Taxodiaceae and Compositae,[62,63,64,65,66,67] while the seeds of Taxodium distichum (L.) Rich.
contain taxodione and quinone methides.[68] (Both quinone methides are relatively stable).
Hazardous effects of phenanthrenequinones to the skin have been described as irritant or as
causing allergic contact dermatitis as early as 1875. H. H. Babcock, a well-known botanist
from Chicago, suffered from severe dermatitis of his hands and face after collecting field-
grown lady’s slippers (Cypripedium calceolus).[69] Similar skin lesions caused by related
Cypripedium and Paphiopedilum species were observed by MacDougal several years
later[70,71] Babcock‘s bad experiences motivated Nestler in Prague to rub leaves and extracts of
different Cypripedium species on the skin of his arms. A month later strong itching
developed, followed by redness and vesicles.[72] Retired individuals who spent all of their
leisure time in breeding orchids developed allergic contact dermatitis as described between
Structure-Activity Relationships in Allergic Contact Dermatitis
37
1957 and 1980[73,74,75] In the eighties several cases of occupational allergic contact dermatitis
have been observed in female employees handling orchids of the genus Cypripedium, Paphio-
pedilum, Cattleya and Cymbidium in orchid nurseries in Lower Saxony (Germany). However,
the sufferers refused any patch testing because they were deeply convinced that “such
beautiful flowers never could be the sources of their skin lesions“. Therefore, only chemical
studies of wild and bred plant material from Germany and the USA were performed revealing
the occurrence of quinonoid constituents in some of these species.[76] In two other nurseries
two cultivators could be tested epicutaneously with leaves, petals and stems as well as with
two isolated quinones giving strong responses. However, due to insufficient plant material
(orchids were extremely expensive at that time) the reacting quinones could not be isolated in
quantities sufficient for structure elucidation.[77] Nestler’s assumption of 1907 that the ‘toxic’
constituent of one of the Cypripedium species might be a red quinone was corroborated more
than 70 years later when the responsible allergen could be identified as 2,8-dimethoxy-7-
hydroxy-1,4-phenanthrenequinone (cypripedin) (compound 5-6, Tab. 5-1).[57]
The most valuable commercial timbers, East Indian rosewood (Dalbergia latifolia) and
African blackwood (Grenadill) (Dalbergia melanoxylon), containing latinone and melatinone,
respectively, have also been blamed for their sensitizing properties.[78]
Perianal contact dermatitis was observed in eight patients in Kenya using maigoya leaves
from Plectranthus barbatus Andr. (family Lamiaceae) as toilet paper.[79] Black bryony
(Tamus communis L.) has since been well known for its irritant properties.[80] Other plants and
timbers of the Combretaceae, Dioscoraceae and Compositae family, containing
phenanthrenequinones and related compounds, have not been described so far for causing skin
reactions. However, Hopff & Schweizer[81] could already demonstrate in early calculations
that some of these phenanthrenequinones are highly reactive in nucleophilic substitution.
Furthermore, there is evidence that phenanthrenes, abundantly occurring as precursors in
distinct plants and timbers, will be oxidized under proper conditions to either ortho- or para-
phenanthrene-quinones.[82]
Structure-Activity Relationships in Allergic Contact Dermatitis
38
5.1.1 The investigated phenanthrene-quinones
Case reports suggest that some or even all of these PACs possess a distinct sensitizing
potency. In order to study the relationship between chemical structure and sensitizing
properties,[83] a number of known, as well as new PACs were synthesized chemically. In a
first step, the Diels-Alder reaction of styrenes and benzoquinones was used, leading to a
number of substituted PACs or the corresponding dihydro components. In an alternative
approach, the well-known photocyclization of stilbenes was exploited to prepare the PACs.
Details of these syntheses have been published recently in a chemical journal.[84] To obtain
more details on structure-activity relationships, guinea pigs were sensitized to determine the
sensitizing capacity of the 26 PACs (four naturally occurring and 22 synthetic PACs).
O
O
R7
R6
R5
R4R3
R1
R2
1
2
3
4
9
5
10
6
7
8
A
B
C
5-1 to 5-23
Scheme 5-1: Substitutions pattern of phenanthrenequinones 5-1 to 5-23
No Phenanthrenequinone (PAC) R1 R
2 R
3 R
4 R
5 R
6 R
7
5-1 1,4-PAC H H H H H H H
5-2 3-Methoxy-1,4-PAC (=
Annoquinone-A)
H OMe H H H H H
5-3 6-Methoxy-1,4-PAC H H H OMe H H H
5-4 7-Methoxy-1,4-PAC H H H H OMe H H
5-5 8-Methoxy-1,4-PAC H H H H H OMe H
5-6 2,8-Dimethoxy-7-hydroxy-1,4-PAC
(= Cypripedin)
OMe H H H OH OMe H
5-7 3,6-Dimethoxy-1,4-PAC H OMe H OMe H H H
5-8 3,7-Dimethoxy-5-hydroxy-1,4-PAC H OMe OH H OMe H H
Structure-Activity Relationships in Allergic Contact Dermatitis
39
(= Denbinobin)
5-9 3,8-Dimethoxy-1,4-PAC H OMe H H H OMe H
5-10 5,8-Dimethoxy-1,4-PAC H H OMe H H OMe H
5-11 6,7-Dimethoxy-1,4-PAC H H H OMe OMe H H
5-12 7,8-Dimethoxy-1,4-PAC H H H H OMe OMe H
5-13 2,7,8-Trimethoxy-1,4-PAC (=
Cypripedin methyl ether)
OMe H H H OMe OMe H
5-14 3,5,7-Trimethoxy-1,4-PAC H OMe OMe H OMe H H
5-15 3,5,8-Trimethoxy-1,4-PAC H OMe OMe H H OMe H
5-16 3,6,7-Trimethoxy-1,4-PAC H OMe H OMe OMe H H
5-17 3,7,8-Trimethoxy-1,4-PAC H OMe H H OMe OMe H
5-18 6,7,8-Trimethoxy-1,4-PAC H H H OMe OMe OMe H
5-19 2,3,7,8-Tetramethoxy-1,4-PAC OMe OMe H H OMe OMe H
5-20 3,5,6,8-Tetramethoxy-1,4-PAC H OMe OMe OMe H OMe H
5-21 8-Benzyloxy-7-methoxy-1,4-PAC H H H H OMe Bn H
5-22 3,6-Dimethoxy-7-hydroxy-9-phenyl-
1,4-PAC (= Latinone)
H OMe H OMe OH H Ph
5-23 6,7-Dimethoxy-9-phenyl-1,4-PAC H H H OMe OMe H Ph
Table 5-1: Substitutions pattern of phenanthrenequinones 5-1 to 5-23
However, such experimental studies in guinea pigs have not been performed so far. Guinea
pigs were sensitized by a modified Freund’s complete adjuvant method (FCA, FCAT,
GPMT)[85] in order to find out which and how many substituents at the carbons of the three
rings of the PAC will influence the sensitizing power of the molecule.
Structure-Activity Relationships in Allergic Contact Dermatitis
40
5.2 Sensitization
A method developed from the Freund’s complete adjuvant test (FCAT) and the guinea pig
maximization test (GPMT); named modified FCAT has been used. Its advantage could be
proven with a large number of compounds, especially in the determination of the sensitizing
capacity of moderate and weak contact allergens. Ten guinea pigs of the Pirbright white strain
were used for each substance. For induction, 15 mg of the PAC was dissolved in 4 mL FCA
test solution and emulsified with 4 mL physiological saline. Then, six intradermal injections
of 0.1 to 0.15 mL of the emulsion were administered in a semicircular arc on the clipped and
shaved shoulder area (4×6 cm) from left to right. Starting on day 1, the procedure was
repeated on the fifth and ninth day, leaving a gap of 2 to 3 cm between the rows of injections.
Each guinea pig received a total of 4.5 mg of the PAC during the entire sensitization
procedure. All sensitization experiments were performed by Prof. B. M. Hausen at the
Department of Dermatology of the University Hospital, Hamburg, Germany.
Elicitation was performed 21 days after induction by open epicutaneous challenge with 0.05
mL of the compound dissolved in acetone in sub irritant molar concentrations and further
dilutions on the left flank of the animals. In case the PAC did not dissolve sufficiently in
acetone, 10 % ethanol, methanol, or ethyl acetate was added. Generally, molar concentrations
(M) were used, starting with 0.3 mol/L and subsequent dilutions, in steps of 1:2 down to
0.0001 mol/L. Readings were performed after 24, 48, and 72 hours. The mean response (mr)
was computed as the quotient of the sum of the reactions observed and the total number of
guinea pigs treated. A mr of 0 to 1 was considered weak, 1 to 2 moderate, and a mr greater
than 2 as strong.
Using the emulsion of FCA and physiological saline but without a PAC, ten guinea pigs were
treated at the same intervals and under the same conditions as described above. One day
before challenge, these animals were tested epicutaneously with 3 different concentrations of
the PACs on the right flank (1.0 mol/L, 0.3 mol/L, and 0.1 mol/L). The results were read after
24 hours. A (+) reaction was considered the threshold of irritancy.
Structure-Activity Relationships in Allergic Contact Dermatitis
41
5.3 Sensitization test results
One quarter of the control animals developed a (+) reaction when challenged with the PACs
in 1.0 mol/L concentration. Thus the safe sub irritant challenge concentration was chosen to
be 0.1 mol/L. The results of the sensitization experiments are summarized in Table 3. The mr
(mean response) given in the following table is the median of all 3 readings observed at a
challenge concentration of 0.003 mol/L. Two PACs (5-21, 5-26) showed a strong sensitizing
capacity, eight a moderate one (5-3, 5-4, 5-5, 5-8, 5-10, 5-12, 5-18, 5-23), and ten a weak
sensitizing capacity (5-1, 5-2, 5-6, 5-7, 5-9, 5-11, 5-15, 5-17, 5-22, 5-25). The five remaining
PACs were nearly (5-14, 5-13, 5-19, 5-20, 5-24) or completely negative (5-16).
Phenanthrenequinone No mr
Strong
8-Benzyloxy-7-methoxy-1,4-PAC (5-21) 2.16
7,8-Dimethoxy-9,10-dihydro-1,4-PAC (5-26) 2.10
Moderate
8-Methoxy-1,4-PAC (5-5) 1.93
7-Methoxy-1,4-PAC (5-4) 1.82
7,8-Dimethoxy-1,4-PAC (5-12) 1.72
5,8-Dimethoxy-1,4-PAC (5-10) 1.63
6,7,8-Trimethoxy-1,4-PAC (5-18) 1.60
6-Methoxy-1,4-PAC (5-3) 1.42
6,7-Dimethoxy-9-phenyl-1,4-PAC (5-23) 1.38
3,7-Dimethoxy-5-hydroxy-1,4-PAC (Denbinobin) (5-8) 1.28
Weak
1,4-Phenanthrenequinone (5-1) 0.88
3-Methoxy-1,4-PAC (Annoquinone-A) (5-2) 0.85
9,10-phenanthrenequinone (5-25) 0.75
Structure-Activity Relationships in Allergic Contact Dermatitis
42
3,8-Dimethoxy-1,4-PAC (5-9) 0.65
6,7-Dimethoxy-1,4-PAC (5-11) 0.60
3,5,8-Trimethoxy-1,4-PAC (5-15) 0.50
3,6-Dimethoxy-1,4-PAC (5-7) 0.43
3,6-Dimethoxy-7-hydroxy-9-phenyl-1,4-PAC
(Latinone)
(5-22) 0.30
3,7,8-Trimethoxy-1,4-PAC (5-17) 0.25
2,8-Dimethoxy-7-hydroxy-1,4-PAC (Cypripedin) (5-6) 0.18
Extremely weak and negative
3,5,7-Trimethoxy-1,4-PAC (5-14) 0.08
3,5,6,8-Tetramethoxy-1,4-PAC (5-20) 0.03
2,7,8-Trimethoxy-1,4-PAC (Cypripedin-methylether) (5-13) 0.02
2,3,7,8-Tetramethoxy-1,4-PAC (5-19) 0.02
7,8-Dimethoxy-1,2-PAC (5-24) 0.02
3,6,7-Trimethoxy-1,4-PAC (5-16) 0.00
Table 5-2: Sensitization test results
The strongest reactions were obtained with PAC (5-21) with two substituents on the remote
carbons of ring C (C-7 and C-8). The other PAC (5-26) with strong sensitizing capacity was
the corresponding dihydrophenanthrenequinone, lacking the 9,10 double bond.
Those PACs with one (5-4, 5-5), two (5-10, 5-12) and in one case three methoxy groups (5-
18) on ring C, ranged among the stronger sensitizers in the moderate group. However, 6-
methoxy-1,4-PAC (5-3), denbinobin (5-8) and 6,7-dimethoxy-9-phenyl-1,4-PAC (5-23)
remained below a mean response of mr =1.5 within the moderate group.
The weak group consisted of the two unsubstituted basic molecules of phenanthrene-
quinones, namely 1,4-PAC (5-1) and 9,10-PAC (5-25). Six of the other seven weak sensitizers
were substituted with a methoxy group at C-3 (5-2, 5-7, 5-9, 5-15, 5-17, 5-22) and one at C-2
Structure-Activity Relationships in Allergic Contact Dermatitis
43
(cypripedin (5-6)), of the quinonoid ring (ring A). 6,7-dimethoxy-1,4-PAC (5-5 to 5-11)
revealed to be an exception.
The extremely weak group (mean response ≤ 0.08) comprised the rest of those PACs that were
substituted at one or both carbon atoms (C-2, C-3) of the quinonoid ring A (5-13, 5-14, 5-19,
5-20, 5-24), including the naturally occurring latinone (5-14) as well as cypripedin methyl
ether (5-13). Although possessing two substituents at C-7 and C-8 of ring C, the ortho-
quinone 24 (1,2-phenanthrenequinone) was the weakest sensitizer. Only one of the 26 PACs
remained completely negative (5-16).
The basic, unsubstituted para- and ortho-phenanthrenequinones 1,4-PAC (5-1) and 9,10-PAC
(5-25) are weak sensitizers (mr = 0.88 and 0.75, respectively). 1,2-PAC, even when
substituted with two methoxy groups at the remote positions of the ring C (5-24) is nearly
negative in its sensitizing properties (mr = 0.02). The most important finding with the 1,4-
PAC-derivatives certifies that substitution of the carbons C-7 and/or C-8 of the ring C, which
are remote positions in the molecule, increases the sensitizing power to moderate (5-4, 5-5, 5-
10, 5-12, 5-18) or even strong (5-21, 5-26). Obviously, two substituents are necessary, but as
expected no great differences are observed between them being either a methoxy or a
benzyloxy group (5-21, 5-26). However, one alkoxy substituent alone at either C-7 or C-8 is
not sufficient enough for a strong sensitizing effect (5-4, 5-5). Introduction of a second
substituent at C-5, as in 5-10 or a third as in 5-18 of ring C, decreases the sensitizing capacity.
With methoxy groups at the quinonoid ring itself (ring A), either in C-3 (e.g. 5-7, 5-14, 5-15,
5-17, 5-20, 5-22) or C-2 (5-6, 5-13) or in both (5-19) the sensitizing capacity weakens
remarkably (mr ≤ 0.5), even if methoxy groups are present at C-7 or C-8 of ring C (5-19, 5-
20).
In contact allergy, side chains of amino acids in proteins function as nucleophiles.
Nucleophilic substitution may occur by an electron-rich nucleophile on an electron-deficient
electrophilic centre such as the double bonds in quinones (Michael-type addition).
Structure-Activity Relationships in Allergic Contact Dermatitis
44
5.4 Quantum mechanical calculations
In an attempt to rationalize the sensitizing capacity of the differently substituted PACs in
terms of chemical reactivity with nucleophilic sites of the proteins, we calculated the LUMO
coefficients at the reactive sites (C-2 and C-3) of a selected number of 1,4-phenanthrene-
quinones using the Spartan Program, Version 1.07 (the AM1 and PM3 methods within the
Spartan program gave nearly identical results). According to the frontier orbital theory,[86]
reactivity is highest with greatest overlap of the corresponding LUMO of the electrophile and
the HOMO of the nucleophile. Thus, with the HOMO of the protein nucleophilic centres to be
assumed constant, chemical reactivity should depend on the size of the LUMO coefficient at
the potential electrophilic centres at C-2 or/and C-3 of the 1,4-phenanthrenequinones. The
LUMO energies and the coefficients at C-2 and C-3 are listed in Table 5-3. The LUMO
calculations were extended to 2-Methoxy-1,4-PAC (5-27) and 9,10-Dihydro-PAC (28), which
had not been included in the sensitizing experiments.
No LUMO energy (eV) LUMO coefficient at C-2 LUMO coefficient at C-3
5-1 1.743 0.0074 0.0075
5-3 2.021 0.0073 0.0053
5-4 2.021 0.0086 0.0069
5-5 2.002 0.0081 0.0070
5-7 1.518 0.0051 0.0038
5-12 1.657 0.0073 0.0073
5-13 1.551 0.0037 0.0086
5-26 1.514 0.0077 0.0085
5-27 - 0.0020 0.0045
5-28 -1.639 0.0107 0.0081
Table 5-3: Calculated LUMO coefficients of 10 phenanthrene-quinones
Structure-Activity Relationships in Allergic Contact Dermatitis
45
In fact, methoxy substitution at C-2 or C-3 (5-2 or 5-27) decreases the coefficients at the
remaining electrophilic centres with respect to the unsubstituted 1,4-phenanthrene-quinone
(1).
Figure 5-1: LUMO of 5-1 and 5-26
In addition, the chemical reactivity is decreased considerably by sterical hindrance at the
neighboring electrophilic site. On the other hand, the LUMO coefficients at C-2 considerably
increased by substitution at C-7 or C-8 (5-4 and 5-5), whereas a methoxy group at C-6 (5-3)
has practically no influence on the size of the LUMO coefficients at C-2, and decreases the
coefficient at C-3. The increase of the coefficients are not influenced by hydrogenation of the
9,10 double bond (5-26) showing a high coefficient at C-3 (5-28).
Figure 5-2: LUMO density of 5-1 and 5-26 (red low, blue high)
Thus, not surprisingly, phenanthrenequinone 5-26 is among the strongest sensitizers. The
correlation and statistical evaluation of the LUMO coefficients at the reactive sites at C-2 and
C-3 and the biologic parameter mr ∆, representing the strength of sensitizing potency, is
shown in Figure 5.3. With the exception of three values, there is a good fit of the data for both
electrophilic centers C-2 and C-3, with regression factors 0.903 and 0.943 respectively. This
correlation is really remarkable considering a complicated biologic effect such as sensitizing
Structure-Activity Relationships in Allergic Contact Dermatitis
46
potency. One reason for this may be the relatively homogeneous series of PACs investigated,
which had comparable polarities and pharmacologic properties.
y = 0,002x + 0,0044
R = 0,903
y = 0,0026x + 0,0024
R = 0,943
0,002
0,003
0,004
0,005
0,006
0,007
0,008
0,009
00,511,522,5
mr ∆
LUMO
LUMO at C-2
LUMO at C-3
Linear (LUMO at C-2)
Linear (LUMO at C-3)
Figure 5-3: Statistical distribution of LUMO coefficients vs. sensitizing capacity
This is a very nice example that theoretically calculated chemical reactivity and the
experimental data of sensitizing capacity are in a very good agreement in a large and
homogeneous group of potential allergens. This approach is very meaningful in the case of the
natural compounds latinone (5-22), cypripedin (5-6), annoquinone-A (5-2), denbinobine (5-8)
and several selected synthetic phenanthrenequinones. It may also be useful in predicting the
sensitizing capacity of potentially allergenic compounds of natural origin such as the
royleanones and tanshinones, which have not been the subjects of sensitization studies so far.
These results might contribute to a better understanding of contact allergy.
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
47
6 Determination of the Absolute Configuration by Quantum
Chemical Calculation of CD Spectra
6.1 Introduction
The knowledge of the stereo structure of biologically active natural products, in particular of
their absolute configuration, is an important precondition, for example, for directed structure-
activity relationship investigation. While the relative configuration can be established
relatively easily spectroscopically (for example by NMR) or by X-ray diffraction, the
assignment of the absolute stereo structure is sometimes a more difficult task, in particular for
novel classes of compounds.
Enantiomers have the same scalar physical characteristics such as melting point, boiling point,
density and chemical reactivity with achiral reaction partners. However, their reactivity with
chiral reagents is different. The biological effect of chiral substances, which are generally
used as herbicides or drugs, also depends on their absolute configurations. Two enantiomers
can differ strongly in their strength and quality of effect depending considerably on the
affinity and selectivity of the active substance to the active site.
The contergan scandal serves as an instructive example. The sedative and antinausea agent,
contergan, with the active substance rac-thalidomide, led to the most serious of medicament
scandals: the contergan disaster.
N
(S)
HN
O
O
O
ON
(R)
HN
O
O
O
O
(S)-Thalidomide (R)-Thalidomide
When taking contergan in the early stage of pregnancy, the children born had a high incidence
of deformities in their limbs (phocomelia). From 1958 to 1961, approximately 10000 children
world-wide were born with some kind of defects. The medicament contergan contained
primarily the active substance rac-thalidomide (Phthalimidoglutarimid), whose R-(+)-
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
48
enantiomer carries the required pharmacological effect, while the (S)-enantiomer is a very
potent teratogen.
Thalidomide was developed in 1956 by a chemist, Dr. Heinrich Mückler, and brought into
trade in October of 1957 by Grünenthal in Stollberg at Aachen. Contergan was removed from
the market in November 1961, when the drugs serious side effects became known.
6.2 Theoretical Principles - Calculation of CD Spectra
Conformation analysis by means of 1H-NMR spectroscopy employs the so called Karplus
rule, which describes the dependence of the vicinal-coupling constant on the torsional angle in
a saturated system. Another method is the measurement of the NOE´s (Nuclear Overhauser
Effect); hereby steric interactions are evaluated. The determination of the absolute
configuration is not always that straightforward.[87]
One of the most efficient if not ideal methods for configurational assignment is the
investigation of circular dichroism (CD). With low experimental demand, two enantiomers
can be easily distinguished by their exactly opposite CD spectra. The assignment of the
absolute configuration of the enantiomers, however, can be complicated, since it quite often
can not be deduced solely from the measured CD spectrum exploiting empirical rules on the
given chromophore (octant rule). Even the widely used exciton chirality method[88,89,90] is
restricted in its application, since it requires the presence of certain functional groups that can
be derivatized and this precondition is not fulfilled in each single case.
The quantum chemical calculation of CD spectra, by contrast, is not limited by any structural
restrictions and thus constitutes a generally applicable procedure. When light passes through
an absorbing optically active substance, not only do the left and the right circularly polarized
rays travel at different speeds, cL ≠ cR, which leads to
λ
L ≠
λ
R, but these two rays are also
absorbed to a different extent; that is,
ε
L ≠
ε
R. The difference
∆ε
≡
ε
L −
ε
R is called circular
dichroism, and all commercially available “dichrographs” the difference in absorption for the
two helical rays is recorded.[89] For a quantitative and standardized description of circular
dichroism we refer to the Lambert-Beer-Bouguer law. If I0 is the intensity of light impinging
on the cell, and I that when leaving, the absorbancy A = log10(I0/I) =
ε
cl; that is, the recorded
signal (A) is proportional to the concentration c and the path length l. If c is given in mol/L
and l in cm, then
ε
is called the molar (decadic) absorption coefficient or molar absorptivity.
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
49
From the definition, it follows that CD can be observed only within absorption bands and
hence it concerns electronic transitions in the VIS or UV range (electronic circular dichroism:
ECD) and vibration bands in IR region (vibrational circular dichroism: VCD). In solution, a
Cotton effect (CE) arises only if the examined substance is chiral. The CD spectra of
enantiomers are mirror-image to the x axis. The rotatory power R0a for a transition 0 → a can
be calculated from the scalar product of the magnetic and electrical transition moment as
follows[91]:
{
}
{}
00 000
ˆˆ
Im Im
aaa aa
Rmm
ψµψ ψ ψ µ
==− (1)
Whereas Im means that one should use only the imaginary part of the complex numbers,
µ
(electrical dipole transition moment) and m (magnetic dipole transition moment) are
operators.
00
0
()
aa
a
eh
p
im E E
µ
=− (2)
Substituting term (2) into term (1) results in:
000 00
00
ˆˆ
Im Im
() ()
aaa aa
aa
eh eh
Rpmpm
im E E im E E
ψψψψ
⎧⎫⎧⎫
==
⎨⎬⎨⎬
−−
⎩⎭⎩⎭
(3)
Term (3) has the advantage that it depends on origin of the advanced wave functions
Ψ
0 and
Ψ
a for the ground and excited states; and p is the operator of impulses. The wave functions
are obtained by CNDO/S-CI calculations. The extension CI means that both the ground state
and the excited state are considered. These calculations were carried out by the program
package BDZDO/MCDSPD developed by Downing and modified by Fleischauer.[92]
For a better demonstration the rotatory power is calculated from the
∆ε
-curves as follows:
00
00
2
0
6.909
() ()
8 1000 aa
A
hc
R
N
νε
εν σ ν
πµ
∆= (4)
Whereas NA represents the Avogadro number and
σ
0a(
λ
) is the Gauss-function:
2
0
1
()
a
a
a
a
e
λλ
σλ π
⎛⎞
−
−⎜⎟
∆
⎝⎠
=−∆ (5)
λa denotes the wavelength of the maximum of ∆ε, and ∆a is the full width at half maximum of
the Gauss-curve in nm.
For the computation of the wave function, it is necessary to know the conformation of the
molecule. At room temperature, the lowest energy conformer can equilibrate with some
higher energy ones, some of which can not be neglected. The change of the relative
orientation of the chromophore has an influence on the CD spectrum. For this reason,
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
50
conformational analysis of the arbitrarily chosen enantiomer is performed first accomplished
by the program Spartan SGI Version 5.3. For this purpose, either a systematic or a Monte-
Carlo search with force field MMFF94 is applied. Subsequently, the heat of formation ∆fH in
kJ/mol is computed semi-empirically by AM1-Model, which can be also gained from the
Spartan program package.
The participation of each conformer is determined by the Boltzmann equation as:
ij
EE
ikT
j
Ne
N
−
−
= (6)
The CD data of all the conformational isomers of the chiral compound up to an energy cut-off
(usually 10−12 kJ/mol) are taken into consideration, because these conformers have more
than 1 % participation to the sum CD spectrum at room temperature (298 K). The CD-spectra
of each conformer is computed and added up by the means of the Boltzmann statistics (6) to
get the final CD-spectrum.
Natural or synthetic
compound
Experimental
UV-Spectrum +Experimental
CD-Spectrum
starting
geometry
conformational
analysis
single
conformational
isomers
(AM1, PM3)
single
UV-spectra
single
CD-Spectra
calculated
CD-spectrum
calculated
UV-spectrum
corrigated
calculated
CD-spectrum
Comparison
UV-Shift
Comparison
Figure 6.1: Diagram the calculation of CD spectrum
When comparing the calculated overall CD spectrum obtained in this manner with the
experimental one, three possibilities arise:
- the two spectra show essentially the same CD curve
- the experimental spectrum shows a virtually opposite CD behavior
- the two spectra have no similarity.
In the first case, the studied compound has the same absolute configuration as the one used for
the calculation. In the second case, it must have the enantiomeric structure. If the two spectra
differ from each other significantly (third case) no statement about the absolute configuration
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
51
can be made. Using this method, the absolute configuration of several natural products such
as Ancistrocladin, Doncophyllin, Palmarumycine, Vismion H etc. have been elucidated.[87]
6.3 Determination of the absolute configuration of an ergochrome
Ergochromes are natural products, which belong to the class of anthranoids and are present in
numerous microorganisms.[93] The first representatives were isolated from an ergot fungus
(Claviceps Purpurea). It has been discovered recently that ergochromes are toxins of food-
fungi, and detailed investigation of their biological activities were initiated. The available
toxicity studies on mice designate this class of compounds as fungus toxin of middle strength.
Antibiotic 5049 (6-1), isolated by our research group from the strain Costus sp. (Costaceae) as
yellow crystals, also belongs to the class of anthranoids, which derives from natural anthracen
compounds.[94]
O
OH O OH
O
OH O OH
ROH2C
CH3
OR
H3C
RO CH2OR
6-1
1
2
3
4a
5
6
7
8a
9
9a
11
12
1'
4'
4
Figure 6.2: Antibiotic 5049, R=OAc
The yellow color of the compound can be explained by both the presence of the chromogenen
tricycle ring system of the anthrone structure and the auxochromen hydroxy groups. The
biosynthetic precursor of this natural product is the secalonic acid A (ergochrome A). All
secalonic acids are dimers of two xanthon monomers connected at the 2,2´-position. The
formation of secalonic acid A can be explained by the enzymatic oxidative ring-opening of
Emodin (a tricyclic quinone derivative). Besides the different position of linkage of the two
monomers, the methyl ester group of secalonic acid A is replaced by methylenacetoxy group.
There are several secalonic acid derivatives known in the literature whose configurations at
position 5,5´, 6,6´, or 10,10´ are different.[95]
The relative stereochemistry of the aliphatic moiety was elucidated by NOE-difference-
spectrum. The strong correlation of the 12-methylene protons with 5-H (+50.3 %) suggested
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
52
the trans-orientation (most likely trans-diaxial) of the substituents at position C-5 and C-10a.
In contrast, a similar strong enhancement of the C-16 protons was observed by irradiation
implying the cis assignment of these positions. Finally, X-ray diffraction confirmed the
former relative conformation.
Figure 6.3: X-ray picture of Antibiotic 5049 (6-1)
The determination of the relative configuration was a prerequisite for the quantum chemical
calculation of the CD-spectra.
The conformational analysis was performed by combining molecular mechanics and semi-
empirical (AM1) methods using Monte-Carlo-Simulation in water-CT, setting 2 as degree of
freedom of the aryl-aryl axes. 8 minimum-energy conformers were found, up to an energetic
cut-off of 11 kJ/mol relatively to the lowest energy conformer whose energy was computed
semi-empirically. The theoretical calculation of the CD-spectra was carried out by the
BDZDO/MCDSPD program package.[92]
The calculated and experimental CD spectra are in a good agreement as shown in Figure 6.4.
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
53
-70
-50
-30
-10
10
30
50
220 240 260 280 300 320 340 360 380 400
λ/nm
∆ε/cm2mol-1
X-ray opt,
Boltzmann
weighting
X-ray
CD measured,
MeOH
Figure 6.4: Comparison of the calculated and measured CD spectra of 6-1
Since this molecule contains not only central but also axial chirality, it should be investigated
which of these chirality elements determine the CD spectrum. In order to find the answer of
this question, CD spectra of several conformers of Antibiotic 5049 (6-1) have been calculated,
which showed that conformers with negative dihedral angle exhibit mirror-image CD-
spectrum.
-80
-60
-40
-20
0
20
40
60
80
150 200 250 300 350 400
λ /nm
∆ε/cm
2
mol
-1
76.65°
-76.06°
-75.79°
78.07°
77.48°
78.75°
75.12°
-77.84°
Figure 6.5: Calculated CD spectra of 8 conformers of 6-1
This experiment points out that the axial chirality plays a decisive role in the CD-spectra.
Since the Cotton-effect at 300 nm (∆ε= 41) derives from the carbonyl n-π* transition, the CD
spectrum of the monomer-moiety (6-2) was calculated in the next step. This monomer has the
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
54
C-10a chirality center, but has no axial chirality element. This way the axial chirality
contribution, which has a dominating effect on the CD spectrum was ruled out.
O
OOH OH
CH3
OAcAcO
1
2
3
45
6
7
8
9
10 11
12
8a
10a
4a
9a
6-2
Figure 6.6: Structure of monomer building block (6-2)
The calculated CD of the monomer 6-2 shows only a negative couplet between 192 nm and
238 nm with a negative peak at 209 nm (∆ε = −33) and a positive one at 290 nm (∆ε= 7),
whereas this curve showed quite different characteristics.
-40
-30
-20
-10
0
10
20
30
150 200 250 300 350 400
λ/nm
∆ε/cm
2
mol
-1
Figure 6.7: Calculated CD of monomer 6-2
6.3.1 Atropismerism
Biphenyls with hindered rotation represent one of the most classical examples of
atropisomerism. They form enantiomers because the two benzene rings are not coplanar and
both rings are substituted unsymmetrically so that the plane passing through the pivot bond
and one of the benzene rings can not be a σ plane. If we consider the conformations of
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
55
biphenyls in more detail, we recognize that there are two diastereomeric conformations
possible.
In biphenyls, the restricted rotation is sustained by bulky atoms in the ortho-positions of the
phenyl rings. Oki (1983)[96] arbitrarily predicted the existence of atropisomerism where
isomers can be isolated for compounds that have a racemization half-life t1/2 > 1000 s (16.7
min). This value does not define the required free energy barrier, which evidently now
depends on temperature; it is 22.3 kcal/mol (93.3 kJ/mol) at 300 K, 26.2 kcal/mol (109.6
kJ/mol) at 350 K and 14.7 kcal/mol (61.5 kJ/mol) at 200 K.[97]
6.3.2 Determination of the rotation barrier of ergochrome (6-1)
Quantum chemical calculations, in particular, ab initio molecular orbital calculations and
density functional calculations, can be applied to furnish data for parameterizing empirical
energy functions (molecular mechanics models). The data related to torsional motions are the
most important and meanwhile these are the most difficult to obtain experimentally, since the
empirical energy function needs to reflect the inherent periodicity. For example, the three-fold
periodicity of rotation about the carbon-carbon bond in ethane may be described by the
functional form:
Eitorsion (ωi) = kitorsion3 (1-cos3 (ωi-ωiequilibrium))
ωiequilibrium is the ideal dihedral angle and kitorsion3 is treated as a parameter. Proper description
of bond torsion also requires, at the very least, a term which are one-fold and two fold
periodic.
Eitorsion (ωi) = kitorsion1 (1-cos1 (ωi-ωieq)) + kitorsion2 (1-cos2 (ωi-ωieq))
+ kitorsion3 (1-cos3 (ωi-ωieq))
kitorsion1 and kitorsion2 are additional parameters. The one-fold term accounts for the difference in
energy between cis and trans conformers, and the two-fold term for the difference in energy
between planar and perpendicular conformers.
In the case of ergochrome (6-1), the so called “coordinate driving” was performed using AM1
semi-empirical model of the Spartan Program. First the dihedral angle was set to 0 ° and then
it was driven from 0 ° to 180 ° in 19 steps. After the 19 geometry optimizations are completed
the energy was calculated semi-empirically to get the following plot:
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
56
-410
-405
-400
-395
-390
-385
-380
-375
-370
-365
-50 0 50 100 150
Dihedral
E (kcal/mol)
Figure 6.8: Coordinate Driving of Dihedral of Ergochrome 6-1
Subtraction of the lowest value from the highest energy value resulted in 37 kcal/mol, which
unambiguously showed the existence of a hindered rotation.
This indicates that in the case of ergochromes the axial chirality must dominate over the
central chirality. In order to clarify this theory, the CD spectra of the biaryl-system 6-3a was
computed. This biaryl-system includes the same chromophore as Antibiotics 5049 (6-1) but
has only the axial chirality element.
OH
OH
O
O
O
O
6-3a
OCH3
OCH3
O
O
O
O
(M)-6-3b
M
Figure 6.9: Structure of biaryl systems 6-3a and 6-3b
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
57
-10
-5
0
5
10
15
200 250 300 350 400
λ/nm
∆ε/cm
2
mol
-1
Boltzmann-Biaryl
Antibiotics 5049
Figure 6.10: Calculated CD of biaryl system 6-3a and measured CD of ergochrome 6-1
This curve has a relatively good agreement with the shape of the CD spectrum of Antibiotic
5049 (6-1), having a positive couplet between 225 and 245 nm with a maximum at 232 nm
(∆ε= 32), a minimum at 256 (∆ε= −1) nm and a positive peak at 317 nm (∆ε= 17). This
proves that the axial chirality element is responsible for the chiroptical activity due to the
hindered rotation around the aryl-aryl axes.
Enantiomeric pure biaryl compound (M)-6-3b was investigated by Bringmann and co-
worker.[98] The chromophore system of 6-3a and 6-3b are very similar, the only difference is
the aliphatic side chain in 6-3a, and that the aryl-hydroxyl group is substituted by a methyl
ether. These differences have only a small influence on the CD-spectra, since the main peaks
belong to the interaction of the benzyl rings and to the n→π* carbonyl transition. The
measured CD spectra of 6-3b shows the same shape of curve as the calculated CD spectra of
6-3a and the measured CD spectra of 6-1, having a strong negative peak around 220 nm (∆ε =
−30), and two smaller positive band at 240 (∆ε= 15) and 290 nm (∆ε= 5). These results also
confirm the previously established theory that the axial chirality dominates over the central
chirality element in the case of these substance classes. These investigations permitted not
only the first attribution of the absolute configuration of compound 6-1, but more importantly,
also showed a way to elucidate the axial configurations of related bisanthrones.[93,94,95]
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
58
6.4 Calculation of the absolute configuration of epieudesmin (6-4)
Lignan 6-4 (epieudesmin) was isolated by Krohn and Riaz from Achillea holosericea.
Members of the genus Achillea are common in the Mediterranean area, Eurasia and North
Africa.[99] Aerial parts of different species of this genus are widely used in folk medicine for
the preparation of herbal teas with antiphlogostic and spasmolytic activity. The extract
exhibits pharmacological activities including anti-bacterial, anti-inflammatory and antiallergic
properties.
O
O
HH
H
H
OCH3
OCH3
H3CO
OCH3
6-4
13
4
6
2
5
Figure 6.11: Structure of lignan 6-4
A large and increasing number of lignans have been characterised and recent progress has
been summarized in the Natural Product Reports.[100] The driving force for many of this work
has been the diversity of biological activities shown by various lignans. Most notably, anti-
tumour activity is displayed by podophyllotoxin. Some of its relatives are of clinical use
against cancer, e.g. etoposide, and lately a new generation of derivatives active against
etoposide resistant cells have been introduced. Anti-HIV, antagonism of viral reverse
transcriptase and PAF, antifungal and immunosuppressive activities are among the other
recorded properties.[100]
While the NMR data revealed the connectivities, the relative configuration of 6-4 was
determined by X-ray analysis, which showed that the chirality centers have either
1R,3S,4R,6S or 1S,3R,4S,6R configuration.
The conformational analysis of the 1R,3S,4R,6S enantiomer was carried out for the calculation
of the CD spectra. The quantum chemical calculation gave 10 conformers whose heat of
formation and CD spectra were computed.
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
59
-80
-60
-40
-20
0
20
40
60
80
190 210 230 250 270 290 310 330 350
λ/nm
∆ε/cm
2
mol
-1
Figure 6.12: Calculated CD spectra of several conformers of lignan 6-4
Using the Boltzmann-weighting, the CD-spectra were added according to their relative
participation. Subsequently the calculated and experimental CD spectra were juxtaposed.
Both curves show a negative peak around 210 nm (∆ε= −69) and a small Cotton-effect for the
1La band around 237 and 240.5 nm (∆ε= 1.5).
-20
-15
-10
-5
0
5
10
15
20
190 240 290 340 390
λ/nm
∆ε/cm
2
mol
-1
calculated
measured
Figure 6.13: Comparison of calculated and experimental CD spectra of lignan 6-4
Comparing these results with data from literature[101] of similar structures (sesamins) with
known absolute configuration, it can be noticed that those substances also show a strong
negative peak around 210 nm, and a positive Cotton-effect for the 1La band near by 237 and
240.5 nm. Since the calculated and measured CD spectra are in good agreement, the structure
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
60
used for the calculation is identical with that of the isolated lignan and hence its absolute
configuration is 1R,3S,4R,6S. Another related structure of lignan 6-4 is syringaresinol, whose
absolute configuration has been established from the X-ray structure of its dibromo
derivative.[102] However, the benzene rings of syringaresinol is substituted by OH-groups in
para position. Those X-ray studies on absolute configuration are in a good agreement with
our quantum chemical calculation results.
6.5 Determination of the absolute configuration of a Xyloketal (6-5)
Xyloketal (6-5) was isolated from a Xylaria species by Yongcheng Lin and coworkers. This
fungus origins from the mangroves of the South China Sea. In the first biological test,
xyloketal (6-5) inhibited acetyl choline esterase at the rate of 1.5×10-6 mol/L (p < 0.01).
Xyloketals represent a novel class of natural products whose common structural element is a
bicyclic ketal. Xyloketal (6-5) is a dimer of two bicyclic ketals, whose two moieties are
connected by a CH2 bridge. It has a rigid structure due to the hydrogen bonding between the
two moieties as shown in Fig. 6.14.
O
OO
O
O
O
O
O
HO
OH
H
H
H
H
1
2
3
4
5
6
7
1´
2´
3´ 4´
5´
6´
7´
6-5
Figure 6.14: Structure of Xyloketal 6-5
The relative configuration of 6-5 was elucidated by X-ray crystallography and NMR-
spectroscopy, which revealed that all the stereogenic centers have either R or S configuration.
Since it has a rigid structure, the number of low energy conformers are limited, which allow
the quantum mechanical calculations of its CD spectrum. Comparison with experimental data
then leads to matching or mismatching curves, allowing the assignment of the absolute
configuration.
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
61
Figure 6.15: Most stabile conformer of Xyloketal 6-5
For the CD calculation, the all-R-enantiomer was taken for conformational analysis. The
calculation showed that the six-membered ring is relatively rigid but the five-membered ring
exhibits some conformational flexibility (Figure 6.13). The calculation resulted in three
conformers whose heat of formation and the CD spectra was computed. Boltzmann weighting
was performed and this Boltzmann weighted curve was compared with the experimental one.
The experimental curve shows a strong negative Cotton effect at 215 nm (∆ε = −15.97) and
two weaker positive peaks at 245 and 270 nm (∆ε = 8.71, 1.58). Although the calculated CD
spectrum has a strong negative band at 220 nm (∆ε = −15.79), it shows only a very weak
positive peak at 245 nm (∆ε = 0.71). Since the most intense Cotton effect around 210−225 nm
belongs to the main transition of the xyloketal (1Ba and 1Bb transition; 215 nm
(∆ε = −15.97)[103] and its calculated and measured parameters showed good agreement, it
could be used for the configurational assignment with a high degree of probability.
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
62
-20
-15
-10
-5
0
5
10
180 230 280 330 380
λ/nm
∆ε/cm2mol-1
Figure 6.16: Boltzmann weighted (triangles) and experimental spectra (circles) of lignan 6-5
Since the experimental CD spectrum had the same negative CD minimum for the 1Ba and 1Bb
transition as the CD curve calculated for the all R-enantiomer, the absolute configurations of
all the stereogenic centers are all-R.
6.6 Determination of the absolute configuration of Ascochin (6-6)
Isocumarine derivate (6-6), named Ascochin, derives from Ascochyta sp. has been isolated by
Krohn and Kock from Meliotus dentatus, ingenious at the coast of the East Sea, Ahrenshoop.
The strain showed fungicide activity in the agar-diffusion test and was cultivated for 28 days
at room temperature on 12 L-Culture biomalt agar.
O
O
CH
3
CHO
OH
HO
1
2
3
4
5
6
7
8
6-6
Figure 6.17: Structure and optimized conformation of ascochin (6-6)
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
63
Since X-ray diffraction does not give any information about the absolute configuration of
ascochin (6-6), it must be determined by calculation and measurement of its CD spectra. For
this purpose, the R enantiomer was arbitrarily selected for quantum chemical conformational
analysis, which resulted in only three optimized conformations, thanks to the rigid structure of
the molecule. The structures of these conformers and the X-ray structure were compared and
showed a meaningful correlation. In all cases, the C-4 methyl is axial thereby indicating the
helicity of the hetero ring. The axial position of this methyl group can be explained by the
position of the formyl group on the peri sp2 carbon atom. To relieve the sterical strain, the C-4
methyl moves out the plane of the hetero ring to the axial position. All single CD spectra thus
obtained were added up by Boltzmann statistics using appropriate heats of formation, to give
the calculated overall CD spectrum for isocumarin 6-6, which showed a strong positive
Cotton-effect at 192 nm (∆ε= 20.75).
-10
-5
0
5
10
15
20
25
150 200 250 300 350 400
λ/nm
∆ε/cm
-2
mol
-1
Figure 6.18: Calculated CD spectra of ascochin (6-6)
Subsequently, the CD spectrum of ascochin (6-6) was measured. The plot showed a strong
positive Cotton-effect at 253 nm (∆ε= 52.44). Compared with the calculated value, a shift of
approximately 60 nm was observed.
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
64
-20
-10
0
10
20
30
40
50
60
200 220 240 260 280 300 320 340 360 380 400
λ/nm
∆ε/cm
2
mol
-1
Figure 6.19: Measured CD of ascochin (6-6)
This shift can be explained by the structure of the molecule 6-6. Since isocumarine 6-6
possesses a multichromophore system, it was assumed that the program was not able to
consider it completely for the calculation. To avoid this problem, the chromophore of
compound 6-6 was modified by catalytic hydrogenation to dihydro-isocumarin 6-7. As
expected, the reaction proceeded diastereoselectively. The hydrogenation gave only the cis
diastereomer under the conditions used (MeOH, Pd/C, H2). On the other hand, dihydro-
isocumarins possess a benzoic ester chromophore, whose chiroptical properties have been
systematically investigated.[104] It was found that the sign of the Cotton effect of n→π* origin
could be safely used for establishing the absolute configuration of the hetero ring of this
chromophore system, the sign of this type of Cotton effect is independent of the substitution
pattern of the aromatic ring system.[105]
For conformation analysis the 3R,4R-dihydro-isocumarin derivate 6-7 was taken. The result
provided three minimum energy conformers, whose CD-spectra were calculated quantum
mechanically. Structure optimization and NMR studies show that C-4 methyl group remained
in the axial position. Thus the helicity of the hetero ring has not changed.
O
OOH
HO
1
2
3
4
5
6
7
8
6-7
CH
3
HO
Figure 6.20: Structure and optimized conformation of dihydro-isocumarine 6-7
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
65
The CD spectra of 6-7 showed a strong negative Cotton-effect at 200 nm (∆ε= −21.40) and
two weak positive ones at 225 nm and 260 nm (∆ε = 6.77, 4.06)
-25
-20
-15
-10
-5
0
5
10
190 210 230 250 270 290 310 330 350 370 390
λ/nm
∆ε/cm
-2
mol
-1
conf 1
conf 2
conf 3
Figure 6.21: Calculated CD spectra of dihydro-isocumarine (6-7) conformers
According to the helicity rule of the chiral benzoic ester chromophore based on the sign of the
n→π* band of conformationally fixed dihydo-isocumarin derivatives,[104] the heterocyclic
ring of 6-7 must adopt a half-chair or a chair conformation, in which the methyl group at C-3
is oriented equatorially. The quantum chemically optimized structure of 6-7 (Figure 6.20)
follows this rule, whereas the hetero ring is in distorted half-chair conformation and the C-3
methyl is equatorial, and the C-4 methyl group has axial position that also determines the
helicity of the hetero ring. Therefore the 3R absolute configuration could also be deduced
from its CD data.[105]
-8
-6
-4
-2
0
2
4
6
8
10
200 250 300 350 400
λ/nm
∆ε/cm
-2
mol
-1
Figure 6.22: Experimental CD spectra of dihydro-isocumarine 6-7
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
66
These results are in good agreement with other dihydro-isocumarin derivates described in
literature.[105] Otherwise the calculated and measured CD spectra are also almost identical.
Compound 6-7 has also a strong negative peak at 218 nm (∆ε= −6.67), and the two weak
positive peaks at 236 nm (∆ε= 5.96) and 266 nm (∆ε= 8.49) were observed. A minimal shift
of 10-20 nm was observed. The conformer, which was chosen for quantum chemical
calculations, has 3R,4R configuration; therefore the absolute configuration of dihydro-
isocumarine (6-7) is 3R,4R also. Since the pyranone-ring of both 6-6 and 6-7 have the same
conformation, and the hydrogenation has not changed the absolute configuration of the stereo
center of 6-6, we can consider that the absolute configuration of isocumarine 6-6 is 4R also.
This is a nice example that if the relative configuration is known, the absolute configuration
could be established both by empirical (helicity) rules and by quantum chemical calculations.
6.7 Determination of the absolute configuration of a metabolic product of
Phomopsis oblonga (6-8) using UV-correction
Phomopsis oblonga (Desm) Trav., a fungus frequently found in the outer bark of healthy
Ulmus sp., particularly wych elm (U. glabra), can invade the phloem of stressed trees,
principally those infected by Ceratocystis ulmi, the causative agent of Dutch elm disease.
Bark beetles of Scolytus sp., the insect vector of the disease, reject P. oblonga-invaded
phloem as being unsuitable for breeding and such trees do not become breed trees. Using a
laboratory bioassay, it was discovered that P. oblonga produces in vitro a number of
boring/feeding deterrents against Scolytid beetles.
Compound 6-8 belongs to a novel class of norsesquiterpene
γ
-lactones to which the name
oblongolide was assigned.[106]
O
O
OH
H
HOH
6-8
1
3
4
5
6
7
89
1a
9a
5a
3a
Figure 6.23 Relative structure of oblongolide 6-8
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
67
After structure elucidation, the X-ray structure was used for conformational analysis, which
resulted in a single low-energy conformer in the calculation.
Figure 6.24: X-ray structure of lactone 6-8
The CD spectrum of this conformer was calculated and it showed a strong negative couplet at
194 nm (∆ε= −22.38), a positive one at 215 (12.85) with a shoulder at 235 nm (7.57).
-25
-20
-15
-10
-5
0
5
10
15
180 200 220 240 260 280 300 320 340 360 380 400
λ/nm
∆ε/cm
-2
mol
-1
Figure 6.25: Calculated CD spectra of lactone 6-8
Since the measured CD spectrum is a mirror image of the calculated one, having a strong
positive transition at 236 nm (∆ε= 3.48), a negative one at 259 (−4.68) with a shoulder at 292
(−2.49), the conformer, which was taken as starting geometry for quantum chemical
calculations, is the mirror image of the natural product: 1a-R,3a-R,5a-R,7-R,9a-S.
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
68
-8
-6
-4
-2
0
2
4
200 220 240 260 280 300 320 340 360 380 400
λ/nm
∆ε/cm
-2
mol
-1
Figure 6.26: Measured CD spectra of lactone 6-8
Jennings, Klyne and Scopes investigated the ORD and CD properties of lactones and
established the “lactone sector rule”, in which the molecules are viewed in the plane of the
lactone group along the bisectix of the O-C-O angle, i.e., the line of the carboxy group and its
attached carbon atom. Thus atoms lying in the back upper right and lower left sectors make
positive contributions to the lactone Cotton effect, while atoms in the back upper left and
lower right sectors make negative contributions.[107] They also studied the class of
sesquiterpene lactones, and found that the compounds with R,R,R,S configuration showed a
positive Cotton effect of the carbonyl n→π* transition at 236 nm. These results are in good
agreement with the measured CD spectra of oblongolide 6-8, since in this case the mirror
image of CD curve is observed. (Fig. 6.26)
The shape of the experimental and the measured curves match also well. (Fig. 6.27) The only
apparent difference is a 40 nm red shift of the measured CD spectrum compared with the
calculated one –a systematic mistake of the quantum chemical calculations. According to the
Brillouin theorem, the excited states and thus the transition energies may be calculated to be
too low when compared to the ground state. This is due to the fact that these calculations can
account for singly occupied excited configurations exclusively. This discrepancy can be
eliminated by an efficient “UV correction”.[87] When calculating the UV spectrum of 6-8
likewise (which should be subject to the same systematic mistake) by using the same
program, the theoretical absorption spectrum was also found to be shifted by about 40 nm
compared to the experimental one, i.e. by virtually the same amount as for the CD spectrum.
A rational scaling of the CD spectrum, by shifting the theoretical CD spectrum by exactly the
same amount, can be established.
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
69
-15
-10
-5
0
5
10
220 240 260 280 300 320 340 360 380 400
λ/nm
∆ε/cm-2mol-1
experimental
calculated
Figure 6.27 Comparison of experimental and calculated CD spectra of lactone 6-8 after UV
correction
This UV-correction can be particularly helpful in those cases in which several CD bands of
different signs follow each other within a smaller spectral range. Taking into consideration
both theoretical[107] and computational results the absolute configuration can be deduced. In
view of this UV-correction, the absolute configuration of 6-8 could be assigned as 1a-S,3a-
S,5a-S,7a-S,9a-R.
6.8 Determination of absolute configuration of Plumericine (6-9) and
Plumenoside (6-10)
Plumericin (6-9) was first isolated by Schönberg and Schmidt 1961[108] from the roots of
Plumeria rubra. Plumenoside was first isolated by Abe from Plumeria acutifolia.[109]
However, their absolute structure has not been elucidated yet. Both plumericin (6-9) and
plumenoside (6-10) belong to Apocynaceae family to the class of iridoids, and show very
good antibiotic, antifungal and antineoplastic activity.
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
70
6-9
O
O
H
CH
3
O
O
OCH
3
O
H
H
H
H
1
2
3
4
5
6
7
89
10
11
12
13
14
15
O
O
H
CH
3
O
O
OCH
3
O
H
H
1
2
3
4
5
6
7
89
10
11
12
13
14
15
HO
6-10
glc
Figure 6.28: Structure of plumericin (6-9) and plumenoside (6-10)
The monoterpenoid natural products are a large, structurally diverse class of compounds that
occur in a wide variety of plant and animal species. A subgroup of this class, the
cyclopentanoid monoterpenes, has been under extensive investigation during the past 30
years.[110,111] Most of the members of this subgroup contain a cyclopentano pyran ring system,
and the name “iridoid” was suggested due to the structural similarity of these compounds to
one of the simplest members, iridodial. Various plants containing iridoids have been used in a
variety of folk medicines for centuries as a bitter tonic, an expectorant, a purgative, and as a
treatment for certain skin diseases. Among the isolated iridoids, demonstrated biological
activities include antibiotic (genepic acid, genipinic acid, plumericin, fulvoplumierin,
udoteatrial), antifungal (plumericin, fulvoplumierin), hypotensive (oleuropein), analgesic
(harpagoside), diuretic (catalposide), antipsychotic (gentianine).[112] All plumericin
derivatives appear in the literature always in the same configuration but there is no reference
about establishing the absolute configuration.
Both plumericin (6-9) and plumenoside (6-10) were isolated from the same plant by Krohn
and Akthar. The relative configuration of 6-9 was established by NMR- and X-ray analysis.
However, plumenoside (6-10) possesses a sugar moiety therefore X-ray diffraction of 6-10
could have provided even the absolute configuration. Unfortunately no single crystal could be
grown. This way the absolute configuration could not be established.
In order to determine the absolute configuration, the X-ray structure of 6-9 was employed as a
basis for quantum chemical calculations. Since plumericin 6-9 has a rigid conformation, it is a
suitable structure for these studies. Comparison of the calculated and experimental CD spectra
gave a very good agreement. There is only a slight shift of 5 nm.
Determination of the Absolute Configuration by Quantum Chemical Calculation of CD Spectra
71
-20
-15
-10
-5
0
5
10
15
180 230 280 330 380
λ/nm
∆ε/cm
2
mol
-1
measured
calculated
Figure 6.29: Calculated and measured CD spectra of plumericin 6-9
The X-ray diffraction revealed that the enone chromophore has planar conformation and
hence it is not inherently chiral. Thus the CD transitions derive from the perturbation effect of
the neighboring stereogenic centers. Both curves show a positive Cotton effect around 191 nm
due to the n→σ* transition of the enone, a negative one at 214.5 nm (∆ε= −5.11) and a
positive one at 237.5 nm (∆ε= 12.16), which arises from the π→π* transition of the enone
chromophore.[113]
For calculation the (1S,5R,9R,10R)-enantiomer was employed and the calculated and
experimental curves are practically the same. Therefore, considering the good agreement of
the calculated and measured CD-spectra the absolute configuration of plumericin (6-9) can be
assigned as 1S,5R,9R,10R. Using this information, the absolute configuration of plumenoside
(6-10) can be designated. On the one hand the optical rotation of 6-9 and 6-10 has the same
sign, and both were found in the same strain. On the other hand, plumenoside (6-10) is a
precursor for the synthesis of plumericin (6-9). After cleavage of the glucose moiety, the ring
closure can be performed by a Michael-addition.[112] This indicates that 6-9 and 6-10 must
have the same absolute configuration.
In the literature several plumericin derivatives are described, whose sign of optical rotation is
the same, but in all cases the mirror image of these substances are presented.[111,108,112,114,110]
The results of this quantum chemical calculation means that the old literature concerning the
absolute structure of other substances of this class must be revised in the light of this recent
finding.
Summary
72
7 Summary
Within the scope of this thesis, the synthesis of biologically active compounds was performed
for a better understanding of the fundamentals of structure-bioactivity relationship, and in
order to develop new agents for a more effective treatment of different plant or human
diseases. These investigations could also open up new opportunities for other applications,
such as targeted drug design and delivery.
Chapter 2 describes the synthesis of benzamide riboside derivatives with potential anitumor
activity. These C-glycosidic analogues of NAD (nicotinamide adenine dinucleotide) (3-1) are
supposed to increase the effect of glucocorticoids, which are administered in the treatment of
malignant lymphoma.[2]
The key step of these syntheses is a metalloorganic coupling-reaction that was carried out by
reacting ribonolactones as electrophiles with organic lithium reagents. The formed tertiary
alcohol was reduced by triethyl silane using borontrifluoride etherate to generate the oxonium
ion. An oxazoline group (2-10) was used as a protecting group for the amide function. In this
manner, the 3-deoxy derivative (2-32) could also be prepared. The coupling of ribonolactone
2-7 with different lithium aryl derivatives (2-33, 2-36, 2-39) proceeded in each case with
yields greater than 75 % over two steps. Investigation on the synthesis of 2-deoxy- (2-53) and
2,3-dideoxy-derivatives (2-54) showed that the presence of a benzyloxy-group in position C-2
of the sugar moiety is indispensable for the coupling.
Clinical trials show that benzamide riboside (2-2) exhibits extreme toxicity against both
human myelogenous leukemia K562 cells and human colon carcinoma HT-29 cells. Test with
the derivatives also showed that both the amide function on the benzene ring and the C-2
hydroxyl group are necessary for the desired antitumor activity.
Chapter 3 The NAD analogue BAD was prepared chemically in order to perform enzyme-
substrate investigations. In cooperation with the Max Plank Institute of Biophysics, in
Frankfurt, X-ray crystallographic study of this complex with reductase will be performed to
investigate the active site of the enzyme. Having information about the active site of the
enzyme can help to decide which part of benzamide riboside (2-2) should be changed to
achieve a better selectivity in tumor cells. Furthermore, this finding can also provide
information about the mechanism of action of reductases. These results could open new ways
for design of biologically more active benzamide riboside analogues.
Summary
73
Chapter 4 deals with the structure activity relationship investigation of a fungicide, named
coniothyriomycin (4-1). Through systematic changing of the substitution pattern of 4-1 it was
shown that fumaric side chain is necessary for the desired antifungal property. The
replacement of the phenyl acetic acid moiety by benzoic acid does not decrease the biological
activity. Electron donating substituents (e.g. OCH3 groups) on the aromatic ring increase the
fungicide effect. Changing the imide function to a hydrazide or thioimide decreases the
activity.
Chapter 5 involves a very nice example that theoretically calculated chemical reactivity
matches the experimental data of sensitizing capacity in a large and homogeneous group of
potential allergens. LUMO coefficients of phenanthrenequinones were calculated semi-
empirically and compared with sensitation tests performed on guinea pigs using Freund´s
complete adjuvant. These results show a very good correlation between chemical reactivity
and sensitizing capacity.
Chapter 6 comprises the determination of the absolute configuration of several natural
compounds whose CD-spectra were calculated theoretically and compared with the
experimental curve. If the two spectra show essentially the same shape of the curve, then the
studied compound has the same absolute configuration as the one used for the calculation. If
the two curves are the mirror image of each other, then the calculated structure is the other
enantiomer. This way the absolute configuration of 6 natural products have been assigned.
The knowledge of the absolute configuration is very important for further structure-activity
relationship investigation.
Experimental Part
74
8 Experimental Part
8.1 Instrumentation
Column Chromatography: Silica gel 60 (230-400 mesh, 0.04-0.063 nm) from Merck.
Thin Layer Chromatography (TLC): Macherey-Nagel, Silica gel 60/F254. The compounds
were visualized by irradiation with 254 or 366 nm light and/or using developing reagents:
¾ 8 % H2SO4 in ethanol
¾ Solution of 10 g Cer-(IV)-sulfate, 25 g Molybdatophosphoric acid and 60 mL cc. H2SO4
in 960 mL water.
IR Spectra: Nicolet FT-IR 510 p. The spectra were measured as KBr pellets or as thin films
of neat compound. The absorptions are given in wave numbers (cm-1).
Optical Rotation: Perkin-Elmer Polarimeter 241 using standard cuvette (d = 10 cm) and Na-
lamp (D-line, λ = 589 nm).
CD-Spectroscopy: JASCO J-715/150S Spectrometer at 25 °C. Temperature control by Peltier
Thermostate, solvent CH3CN. The spectra were recorded by Dr. Tibor Kurtán, Department of
Organic Chemistry, University of Debrecen, Hungary.
Mass Spectra: Fison MD 800. Relative intensity is related to basis peak.
NMR spectra: BRUKER ARX 200, AMX 300, ARX 500. All spectra were recorded at room
temperature (RT). 1H-NMR and 13C-NMR spectra the chemical shift values are given in ppm
relative to SiMe4. The resonance multiplicity is described as s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet). Broad resonances are indicated broad (br).
Melting Point: Gallenkamp Melting point Apparatus, using one-side open capillary.
Purification of Solvents: Purification of solvents was performed by standard methods.[115,116]
Experimental Part
75
Program for CD-Spectra calculation: BDZDO/MCDSPD Program packet Version Z-07A
from J. W. Downing, Department of Chemistry and Biochemistry, University of Colorado,
Boulder, USA; modified by J. Fleischauer, W. Schlecker und B. Kramer or DZDO Program
Version 4.23, ZDO Program Version of 22.06.2001.
Program to calculate the conformers: Spartan SGI Version 5.1.3; Wavefunction Inc.,
Irvine, USA.
8.2 Experimental Part to Chapter 2
2-(3-Bromphenyl)-4,4-dimethyl-2-oxazoline (2-10)[27]
Br
O
N
To a solution of 2-amino-2-methyl-1-propanol (14.4 mL, 0.15 mol) in abs. dichloromethane
(30 mL) m-bromo benzoyl chlorid (10 mL, 0.075 mol) in dichloromethane (30 mL) was
dropped slowly under argon atmosphere at 0 °C. The reaction mixture was stirred for 2 h at
25 °C, and white crystals precipitated. The crystals were filtered off and washed with water.
The filtrate was evaporated and the precipitated crystals were filtered off, and washed with
water.
After drying this intermediate thionyl chloride (14.6 mL, 0.2 mol) was added slowly.
Formation of a gas was observed. The reaction mixture was then poured into diethyl ether and
the precipitated crystals were filtered off. The hydrochloride salt was neutralized by the
addition of 20 % aqueous NaOH solution and extracted three times with ether. The organic
phases were dried over Na2SO4. After evaporation of the solvent, the product could be
isolated as a colorless oil.[27] (TLC: petrol ether/ethyl acetate: 7: 3). Yield: 11.8 g (75 %).−
Experimental Part
76
Methyl-D-ribofuranoside (2-4)[24]
O
OHHO
HO
OMe
D-ribose (25.0 g, 0.17 mol) was dissolved in dry methanol (450 mL) at 0 °C and cc. sulfuric
acid (2.5 mL) was added. After stirring for 16 h at 4 °C, the reaction mixture was neutralized
by the addition of ion-exchange resin (Amberlite XAD-4 stored over saturated NaOH-
methanol solution). After filtration, the solvent was evaporated under vacuum. The viscous oil
was dried under high vacuum. Yield: 26.8 g (98 %).−
Methyl-2,3,5-tri-O-benzyl-D-ribofuranoside (2-5)[25]
O
OBnBnO
BnO
OMe
Methyl riboside (2-4) (25.0 g, 0.162 mol) was dissolved in abs. DMF (300 mL), and 60 %
sodium hydride (35.0 g, 0.875 mol) was added. Formation of hydrogen gas was observed;
then tetrabutylammonium iodide (18.0 g, 0.016 mol) was added. After half an hour, benzyl
bromide (60 mL, 0.875 mol) was added dropwise at 0 ° and the reaction mixture was kept at
50 °C overnight. (TLC monitoring: petrol ether: ethyl acetate = 3:1.) The reaction mixture
was poured into 1 L of ice water and extracted once with diethyl ether and three times with
dichloromethane. The combined organic phases were dried over Na2SO4 and evaporated at
reduced pressure. After column chromatography (first petrol ether, then petrol ether/ethyl
acetate 3:1) 59.9 g (84 %) benzyl ether (2-5) was obtained.−
1H-NMR (200 MHz): δ = 3.31 (s, 3H, OCH3), 3.49 (d, J4,5a = 3.3 Hz, Jgem = 10.6 Hz, 1H, 5a-
H), 3.59 (dd, J4,5b = 3.3 Hz, Jgem = 10.6 Hz, 1H, 5b-H), 3.83 (d, J = 4.6 Hz, 1H, 3-H), 4.02
(dd, J1,2 = 4.6, J2,3 = 6.9 Hz, 1H, 2-H), 4.34 (m, 1H, 4-H), 5.21 (d, J1,2 = 6.5 Hz, 1H, 1-H),
4.39-4.64 (m, 6H, 3 CH2Ph), 7.26 (m, 15H, ArH).–
Experimental Part
77
2,3,5-Tri-O-benzyl-D-ribofuranoside (2-6)[25]
O
OBnBnO
BnO
OH
Benzyl ether (2-5) (55.0 g, 0.13 mol) was dissolved in dioxane (300 mL) and aqueous 1N HCl
(220 mL). The reaction mixture was heated under reflux for 3 hours. After cooling to room
temperature, the reaction mixture was neutralized by the addition of aqueous saturated
NaHCO3 solution. The solvents were evaporated at reduced pressure, and the residue was
dissolved in dichloromethane. The organic phase was washed with water, and the combined
organic phases were dried over Na2SO4, and evaporated at reduced pressure. A yellow oil was
obtained, which was purified by column chromatography (petrol ether/ethyl acetate: 2:1) to
yield 42.5 g of 2-6 (84.4 %).−
1H-NMR (200 MHz): δ = 3.41 (d, J4,5a = 3.5 Hz, Jgem = 10.4 Hz, 1H, 5a-H), 3.50 (m, 1H, 3-
H), 3.60 (dd, J4,5b = 3.7 Hz, Jgem = 10.4 Hz, 1H, 5b-H), 3.63 (m, 1H, 2-H), 4.39 (m, 1H, 4-H),
5.23 (d, J1,2 = 6.4 Hz, 1H, 1-H), 4.39-4.64 (m, 6H, 3 CH2Ph), 7.26 (m, 15H, ArH).–
2,3,5-Tri-O-benzyl-D-1,4-ribonolactone (2-7)[26]
O
OBnBnO
BnO
O
Acetic acid anhydride (90.0 mL) and dimethyl sulfoxide (400.0 mL) were stirred under argon
atmosphere for 1 hour. 2,3,5-Tri-O-benzyl-D-ribofuranoside 2-6 (42.0 g, 0.103 mol) was then
added and the reaction mixture was stirred at room temperature overnight, poured into ice
water (250.0 mL) and stirred for another hour. The reaction mixture was extracted with
dichloromethane and the organic phase was dried over Na2SO4. After filtration, the solvent
was evaporated at reduced pressure to yield a yellowish-orange oil, which was purified by
column chromatography (petrol ether/ethyl acetate: 4:1). The product 2-7 (40.0 g, 94 %) was
dried under vacuum, and crystallized from ethanol. mp.: 34 °C.−
Experimental Part
78
1H-NMR (200 MHz): δ = 3.63 (dd, 1H, J4,5a = 2.7 Hz, Jgem = 11 Hz, 1H, 5a-H), 3.69 (dd, J4,5b
= 2.8 Hz, Jgem = 11 Hz, 1H, 5b-H), 4.21 (m, 1H, 3-H), 4.49 (m, 1H, 2-H), 4.55 (m, 1H, 4-H),
4.61-4.85 (m, 6H, 3 CH2Ph), 7.26 (m, 15H, ArH).–
13C-NMR (200 MHz): δ = 69.3 (CH2, C-5), 72.8 (OCH2), 73.2 (OCH2), 74.04 (OCH2), 74.4
(CH, C-3), 75.9 (CH, C-4), 82.3 (CH, C-2), 128.1-129.01 (ArH), 137.5 (q, ArH), 137.7 (q,
ArH), 137.8 (q, ArH), 174.4 (C=O).–
2-[3-(2,3,5-Tribenzyl-
β
-D-ribofuranosyl)-phenyl]-4,4-dimethyl-2-oxazoline (2-14)[64]
O
OBnBnO
BnO
O
N
A solution of bromo oxazoline (2-10) (4.55 g, 18 mmol) in anhydrous THF (50 mL) was
treated under nitrogen at −85 °C within 10 min with a solution of n-BuLi (12 mL, 1.5 M) in
hexane. After 20 min at −85 °C, a solution of the lactone (2-7) (5.00 g, 12 mmol) in THF (30
mL) was added over 30 min and stirred for an additional 1 h and then warmed over 2 h to −30
°C (TLC control). The reaction was then quenched by the addition of water (30 mL) and
extracted with diethyl ether (100 mL). The organic phase was dried with Na2SO4 and
evaporated under reduced pressure to afford a slightly red oil.
The residue was dissolved in CH2Cl2 and treated at −78 °C with boron trifluoride diethyl
etherate (4.5 mL, 35.43 mmol) and triethylsilane (5.7 mL, 35.78 mmol). The reaction mixture
was stirred for 1 h at −78 °C, warmed overnight to 10 °C, neutralized by the addition of a
saturated aqueous sodium hydrogencarbonate solution (ca. 15 mL), and extracted with CH2Cl2
(100 mL). The solution was dried over Na2SO4 and purified by column chromatography
(hexane/5 % ethyl acetate) to afford 2-14 as a yellow oil (6.02 g, 87 %).−
IR (film): 3063 (aromatic), 2967, 2925 (CH3, CH2), 2892, 2867 (CH), 1779, 1651 (C=N),
1604 (aromatic), 1454, 1363, 1355, 1267, 1124 (ether), 1121, 1092, 1062, 1028, 994, 972,
911, 806.–
Experimental Part
79
UV: 208 (4.64), 239 (sh, 4.06).–
1H-NMR (300 MHz): δ = 1.37 (s, 6H, 2 CH3), 3.66 (ddd, J4,5a = 3.9 Hz, J4,5b = 4.0 Hz, Jgem =
10.4 Hz, 2H, 5-H), 3.83 (dd, J2,3 = 5.2 Hz, J1,2 = 6.5 Hz, 1H, 2-H), 4.02 (dd, J2,3 = 5.0 Hz, J3,4
= 4.0 Hz, 1H, 3-H), 4.05 (2 s, 2H, CH2), 4.34 (m, 1H, 4-H), 5.05 (d, J1,2 = 6.5 Hz, 1H, 1-H),
7.31 (m, 16H, ArH), 7.52 (d, J5,6 = 7.8 Hz, 1H, 6´-H), 7.88 (d, J4,5 = 7.8 Hz, 1H, 4´-H), 7.99
(s, 1H, 2´-H).–
13C-NMR (300 MHz): δ = 29.01 (CH3), 29.02 (CH3), 68.17 (CH), 70.91 (CH2, 5-C), 72.55
(OCH2), 72.88 (OCH2), 74.04 (OCH2), 78.03 (CH), 79.64 (CH2, oxaz), 82.47 (CH), 82.87
(CH), 84.32 (CH), 126.64 (ArC), 128.19 (ArC), 128.26 (ArC), 128.30 (ArC), 128.35 (ArC),
128.45 (ArC), 128.54 (ArC), 128.62 (ArC), 128.74 (ArC), 128.87 (ArC), 128.91 (ArC),
128.96 (ArC), 129.10 (ArC), 129.16 (q, ArC), 129.28 (ArC), 129.82 (ArC), 138.28 (q, ArC),
138.53 (q, ArC), 138.78 (q, ArC), 141.33 (q, ArC), 162.63 (q, oxaz-C).–
Anal. Calcd. for C37H39NO5: (577.28): C: 76.92, H: 6.80, N: 2.42;
Found.: C: 76.86, H: 6.77, N: 2.40.−
3-(2,3,5-Tribenzyl-
β
-D-ribofuranosyl)benzoic acid (2-16)[64]
O
OBnBnO
BnO
COOH
A solution of oxazoline (2-14) (25.0 g, 47.7 mmol) in nitromethane (71 mL) was treated with
methyl iodide (35 mL) and refluxed for 22 h. The solvents were evaporated under reduced
pressure, and the residue was dissolved in methanol (200 mL) and 20 % aqueous KOH (250
mL) and refluxed for 44 h. Half of the solvent was removed under reduced pressure, and the
mixture was then neutralized by the addition of HCl and extracted three times with ethyl
acetate. The organic phase was dried over Na2SO4 and evaporated under reduced pressure to
afford the crude acid 2-16 (35.0 g, 76 %) that could be used for the next reaction without
purification. [α]d23 = 12.41 ° (c = 0.6 in methanol).−
Experimental Part
80
IR (film): 3040, 2880, 2920, 1690, 1610, 1580, 1500, 1460, 1410, 1360, 1200, 1100, 820,
750, 700.–
UV: 206 (4.18), 226 (sh, 3.76), 275 (2.79).–
1H-NMR (300 MHz): δ = 3.64 (ddd, J4,5a = 3.7 Hz, J4,5b = 3.8 Hz, Jgem = 10.4 Hz, 2H, 5-H),
3.81 (dd, J2,3 = 4.0 Hz, J1,2 = 6.4 Hz, 1H, 2-H), 4.02 (dd, J2,3 = 5.0 Hz, J3,4 = 4.0 Hz, 1H, 3-H),
4.37 (m, 1H, 4-H), 4.41-4.67 (3 AB-systeme, 2H, 3 CH2Ph), 5.07 (d, J1,2 = 6.8 Hz, 1H, 1-H),
7.16-7.38 (m, 16H, ArH), 7.64 (d, J5,6 = 7.5 Hz, 1H, 6´-H), 8.01 (d, J4,5 = 7.5 Hz, 1H, 4´-H),
8.19 (s, 1H, 2´-H).–
13C-NMR (300 MHz): δ = 69.85 (CH2, 5-C), 73.52 (OCH2), 73.95 (OCH2), 75.14 (OCH2),
78.03 (CH), 82.35 (CH), 82.76 (CH), 84.23 (CH), 126.62 (ArC), 128.21 (ArC), 128.27 (ArC),
128.31 (ArC), 128.37 (ArC), 128.44 (ArC), 128.53 (ArC), 128.64 (ArC), 128.75 (ArC),
128.88 (ArC), 128.89 (ArC), 128.95 (ArC), 129.11 (ArC), 129.26 (ArC), 129.80 (ArC),
130.08 (q, ArC), 138.27 (q, ArC), 138.55 (q, ArC), 138.80 (q, ArC), 141.35 (q, ArC), 169.33
(q, C=O).−
Anal. Calcd. for C33H32NO6: (524.6): C: 75.55, H: 6.15;
Found.: C: 75.38, H: 6.21.−
3-(2,3,5-Tribenzyl-
β
-D-ribofuranosyl)benzamide (2-18)
O
OBnBnO
BnO
CONH2
A solution of the acid (2-16) (35.0 g, 66.8 mmol) in thionyl chloride (100.0 mL) was treated
with ten drops of DMF and refluxed for 2-3 h. The residue of thionyl chloride was distilled
off. The brown oil was dissolved in CH2Cl2 and the solution was added dropwise to a cold
concentrated aqueous solution of ammonia (300 mL) and then extracted with water and
dichloromethane. The organic phase was dried over Na2SO4, evaporated under reduced
Experimental Part
81
pressure, and purified by column chromatography (first ethyl acetate then CH2C12/5 %
MeOH) to afford the amide 2-18 (4.61 g, 97 %): mp 98 °C (diethyl ether/pentane); [α]d23 =
−48 ° (c = 1.2 in CCl4) .−
IR (film): 3360, 3210 (NH2), 2940, 2880, 1670 (C=O), 1640 (amid II), 1610, 1590, 1500,
1460, 1390, 1140, 1100 (ether), 820 (aromatic), 750, 700.–
1H-NMR (300 MHz): δ = 3.64 (ddd, J4,5a = 3.5 Hz, J4,5b = 3.8 Hz, Jgem = 10.4 Hz, 2H, 5-H),
3.70 (q, 1H, 4-H), 3.82 (dd, J2,3 = 5.1 Hz, J1,2 = 6.4 Hz, 1H, 2-H), 4.05 (t, 1H, 3-H), ), 4.43-
4.63 (3 AB-systems, each 2H, 3 CH2Ph), 5.06 (d, J1,2 = 6.4 Hz, 1H, 1-H), 6.01 (s, 2H, NH2),
7.15-7.36 (m, 16H, ArH), 7.51 (d, J5,6 = 7.5 Hz, 1H, 6´-H), 7.76 (d, J4,5 = 7.5 Hz, 1H, 4´-H),
7.78 (s, 1H, 2´-H).–
13C-NMR (300 MHz): δ = 70.29 (CH2, 5´-C), 71.99 (OCH2, CH2Ph), 72.30 (OCH2, CH2Ph),
73.48 (OCH2, CH2Ph), 77.19 (CH, 3´-C), 81.81 (CH, 4´-C), 82.09 (CH, 1´-C), 83.66 (CH, 2´-
C), 124.57-133.39 (19 ArC), 137.53 (q, ArC), 137.98 (q, ArC), 140.94 (q, ArC), 169.31 (q,
C=O).–
Anal. Calcd. for C33H33NO5: (523.6): C: 75.70, H: 6.35, N: 2.67;
Found: C: 75.42, H: 6.32, N: 2.55.−
3-(
β
-D-Ribofuranosyl)-benzamide (2-2)[64]
O
OHHO
HO
CONH2
A solution of the benzyl ether (2-18) (2.00 g, 3.82 mol) in THF (50 mL) and methanol (2 mL)
was hydrogenated at atmospheric pressure (200 mg 10 % palladium/charcoal, 18 h stirring).
The catalyst was filtered off over Celite, the solvent was evaporated under reduced pressure.
The product was purified by column chromatography (15 % CH3OH/CH2Cl2) to afford 2-18
Experimental Part
82
as a colorless oil (895 mg, 93 %): [α]d23 = −28 ° (c = 0.7 in methanol) and open chained side
product 2-19 as white powder.–
IR (film): 3360 (OH), 2925, 1663 (C=O), 1606, 1581, 1397, 1115, 1072, 1051.–
UV: 207 (3.25), 223 (sh, 2.93), 274 (2.17).–
1H-NMR (D6-DMSO, 300 MHz): δ = 3.55, 3.59 (2 dd, J4,5 = 4.4 Hz, Jgem = 11.7 Hz, 2H, 5-H),
3.90 (dd, J2,3 = 5.4 Hz, J1,2 = 7.1 Hz, 1H, 2-H), 3.83 (q, 1H, 4-H), 3.90 (t, 1H, 3-H), 4.60 (d,
J1,2 = 7.1 Hz, 1H, 1-H), 7.36 (s, 2H, NH2), 7.41 (t, J = 7.7 Hz, 1H, 5´-H), 7.56 (d, J5,6 = 7.7
Hz, 1H, 4´-H), 7.77 (d, J4,5 = 7.7 Hz, 1H, 6´-H), 7.96 (s, 1H, 2´-H).–
13C-NMR (D6-DMSO, 300 MHz): δ = 62.10 (CH2, 5-C), 71.47 (CH), 77.60 (CH), 82.89
(CH), 85.31 (CH), 125.52 (ArC), 126.46 (ArC), 127.99 (ArC), 129.21 (ArC), 134.17 (q,
ArC), 141.55 (q, ArC), 168.09 (q, C=O).–
Anal. Calcd. for C12H15NO5: (253.25): C: 56.91, H: 5.97, N: 5.53;
Found: C: 55.87, H: 6.44, N: 5.45.−
(2S, 3S, 4R)-3-(2,3,4-Tetrahydroxypentyl)benzamide (2-19)
HO
H
OH
HHO HO
H
CONH2
mp.: 134 °C, white powder.−
IR (KBr): 3303 (OH), 1643, 1607, 1577, 1387, 1070, 1016, 767.–
UV: 206 (3.22), 229 (2.72), 275 (1.90).–
1H-NMR (D6-DMSO, 300 MHz): δ = 2.59 (dd, Jgem = 13.8 Hz, J1b,2 = 9.4 Hz, 1H, 1b-H), 2.94
(dd, J1a,2 = 2.3 Hz, Jgem = 13.8 Hz, 1H, 1a-H), 3.42-3.90 (m, 9H, 2, 3, 4, 5-H, 4 OH), 7.36 (s,
Experimental Part
83
2H, NH2), 7.38 (t, J = 7.5 Hz, 1H, 5´-H), 7.75 (d, J5,6 = 7.6 Hz, 1H, 4´-H), 7.84 (d, J4,5 = 7.6
Hz, 1H, 6´-H), 7.99 (s, 1H, 2´-H).–
13C-NMR (D6-DMSO, 300 MHz): δ = 13.16 (CH), 37.30 (CH2), 62.46 (CH2), 72.09 (CH),
73.66 (CH), 123.87 (ArC), 126.76 (ArC), 127.97 (ArC), 131.72 (ArC), 133.01 (q, ArC),
139.73 (q, ArC), 167.68 (q, C=O).–
Anal. Calcd. for C12H17NO5: (255.27): C: 56.46, H: 6.71, N: 5.49;
Found: C: 55.16, H: 6.80, N: 5.36.−
D-Xylono-1,4-lactone (2-21)[33]
OO
OH
HO
OH
A solution of D-xylose 2-20 (30 g, 0.2 mol) and water (100 mL) was cooled to 0 °C and
K2CO3 (34 g, 0.232 mol) was added in small portions. Bromine (12 mL, 0.216 mol) was
added dropwise. The solution was kept at 0 °C for a half an hour and warmed up to room
temperature overnight.
The reaction mixture was worked up by the addition of formic acid. Evaporation of the
solvent resulted in white crystals (a mixture of D-xylono-1,4-lactone and inorganic salts,
which was used for the next step without further purification).−
2,3,5-Tri-O-benzoyl-D-1,4-xylonolactone (2-22)[34]
OO
OBzl
BzlO
OBzl
A reaction mixture of benzoyl chloride (32 mL, 0.13 mol), pyridine (40 mL), and chloroform
(32 mL) was cooled with ice to 0 °C, and xylonolactone (2-21) (5.0 g, 0.043 mol) was added
slowly. After 1 hour the reaction mixture was diluted with chloroform and extracted with
saturated aqueous NaHCO3 solution. The organic phase was dried over MgSO4 and the
Experimental Part
84
solvents were evaporated. The product was recrystallized from diethyl ether to yield 2-22 (9.0
g, 60 %), mp.: 156-158 °C, [α]d23= 24 ° (in aceton).−
IR (KBr): 3062, 2958, 1797 (C=O), 1724 (C=O), 1452 (ν C=C), 1272 (C-O(C), 1114 (C-
O(C), 707 (γ =CH).−
1H-NMR (CDCl3, 200 MHz): δ = 4.72 (m, 2H, 5a,b-H), 5.40 (m, 1H, 3-H), 6.15 (m, 1H, 4-H),
6.19 (m, 1H, 2-H), 7.36-7.67 (m, 9H, ArH), 7.98-8.14 (m, 6H, ArH) (m, 9H, ArH).–
13C-NMR (CDCl3, 200 MHz): δ = 62.0 (CH2, C-5), 71.8 (CH, C-3), 73.4 (CH, C-4), 75.9
(CH, C-2), 128.3-130.6 (ArC), 134.1 (ArC), 134.4, (ArC), 134.6 (ArC), 165.6 (C=O), 169.0
(C=O, C-1).−
3-Deoxy-2,5-di-O-benzoyl-D-1,4-xylonolactone (2-24)
OO
OBzl
BzlO
2,3,5-Tri-O-benzyl-D-1,4-xylonolactone (2-22) (10.0 g, 0.0217 mol) was dissolved in ethyl
acetate (75 mL) and triethylamine (2 mL). The solution was hydrogenated overnight using
10 % palladium/charcoal. The reaction mixture was filtered over Celite and washed first with
aqueous sat. NaHCO3 solution, and then with 4M HCl. The organic phase was dried over
MgSO4 and the solvent was evaporated to afford 2-24 (5.0 g), which was recrystallized from
ethanol. Yield 67 %, mp.: 135-137 °C, [α]d23 = −149 ° (in CH2Cl2).−
IR (KBr): 3059, 2977, 1784 (C=O), 1727 (C=O), 1452 (ν C=C), 1272 (C-O(C), 1114 (C-
O(C), 707 (γ =CH).−
1H-NMR (CDCl3, 200 MHz): δ = 2.25-2.32 (o, 1H 3a-H), 2.35-2.42 (o, 1H, 3b-H), 4.55 (dd,
J1= 3 Hz, J2= 10 Hz, 1H 5a-H), 4.71 (dd, J1= 5.5 Hz, J2= 7 Hz, 1H, 5b-H), 4.94 (m, 1H, 4-H),
5.80 (t, J= 9 Hz, 1H, 2-H), 7.45-7.53 (m, 4H, ArH), 7.59-7.68 (m, 2H, ArH), 8.08-8.13 (m,
4H, ArH).–
Experimental Part
85
13C-NMR (CDCl3, 200 MHz): δ = 31.4 (CH2, C-3), 65.3 (CH2, C-5), 68.9 (CH, C-4), 74.8
(CH, C-2), 128.9-134.2 (ArC), 165.3 (C=O), 166.0 (C=O), 171.6 (C=O, C-1).–
Mass calcd.: 340.09;
MS (EI-CI) m/z %: 341.2 [M+] (63%), 218 [M+−OBzl], 105, 77, 51.−
Anal. Calcd. for C19H20O4 (340.09) C: 67.05, H: 4.74;
Found: C: 66.53, H: 4.69.−
3-Deoxy-2,5-dibenzyl-D-1,4-xylonolactone (2-26)
OO
OBn
BnO
3-Deoxy-2,5-di-O-benzoyl-D-1,4-xylonolactone (2-24) (4.3 g 12.6 mmol) was suspended in
methanol (120 mL) and NaOMe (3 mL) solution was added. After 1 hour the solution was
neutralized by the addition of Amberlite IR-120 on-exchange resin, evaporated, and dried at
reduced pressure. The residue was dissolved in abs. DMF and NaH (0.912 g, 38.0 mmol) was
added. After ½ h, tetrabutylammonium iodide (1.22 g, 3.8 mmol) and benzyl bromide (4.5
mL, 38.0 mmol) was added slowly and stirred at room temperature overnight.
The reaction mixture was poured into ice-water and extracted with dichloromethane. The
organic phase was dried over MgSO4 and the solvent evaporated. The residue was purified by
column chromatography (PE: EE: 3:1) to yield 2-26 (1.81 g, 46 %) as a yellow oil. [α]d23=
−5.61 ° (in CH2Cl2).−
IR (film): 3064, 2925, 1789 (C=O), 1454 (ν C=C), 1272 (C-O(C)), 1122 (C-O(C), 698 (γ
=CH).−
1H-NMR (CDCl3, 200 MHz): δ = 2.08-2.27 (o, 1H 3a-H), 2.47-2.60 (o, 1H, 3b-H), 3.68 (d,
2H, 5a,b-H), 4.31 (t, J= 9 Hz, 1H, 2-H), 4.54 (m, 1H, 4-H), 6.41 (s, 2H, ArCH2), 4.90 (dd, J1=
12 Hz, J2= 32 Hz, 2H, ArCH2), 7.39 (m, 10H, ArH).–
Experimental Part
86
13C-NMR (CDCl3, 200 MHz): δ = 32.0 (CH2, C-3), 71.4 (CH2, C-5), 72.7 (OCH2), 73.6 (CH,
C-4), 73.9 (OCH2), 76.1 (CH, C-2), 128.1-128.9 (ArC), 137.4 (ArC), 138.8 (ArC), 175.0
(C=O, C-1).–
Mass calcd.: 312.14;
MS (EI-CI): 313 [M+1] (24 %), 223.2, 181.2, 133.1, 57.1.−
Anal. Calcd. for C19H20O4: (312.14): C: 67.05, H: 4.74;
Found: C: 67.01, H: 4.78.−
2-[3-(3-Deoxy-2,5-dibenzyl-
β
-D-ribofuranosyl)-phenyl]-4,4-dimethyl-2-oxazolin (2-29)
O
OBn
BnO
O
N
A solution of bromo oxazoline (2-10) (834 mg, 3.36 mmol) in anhydrous THF (20 mL) was
treated under nitrogen atmosphere at −85 °C within 10 min with a solution of n-BuLi (2.2 mL,
1.6 M) in hexane. After 20 min at −85 °C a solution of the lactone (2-26) (700 mg, 2.24
mmol) in THF (30 mL) was added over 30 min and stirred for an additional 1 h and then let to
warm up within 2 h to −30 °C (TLC control). The reaction was then quenched by the addition
of water (10 mL) and extracted with diethyl ether (30 mL). The organic phase was dried with
Na2SO4 and evaporated under reduced pressure to afford a slightly red oil.
The residue was dissolved in CH2Cl2 and treated at −78 °C with boron trifluoride diethyl
etherate (0.84 mL, 6.67 mmol) and triethylsilane (1.06 mL, 6.67 mmol). The reaction mixture
was stirred for 1 h at −78 °C, warmed overnight to 10 °C, neutralized by the addition of a
saturated aqueous sodium hydrogencarbonate solution (ca. 15 mL), and extracted with CH2Cl2
(100 mL). The solution was dried over Na2SO4 and purified by column chromatography
(hexane/ethyl acetate: 3: 1) to afford 2-29 as a colorless oil (700 mg, 67 %), [α]d20 = −93 ° (in
CH2Cl2).−
Experimental Part
87
IR (film): 3031, 2927, 1722 (ν C=N), 1454 (ν C=C), 1272 (C-O(C)), 1106 (νas C-O-C), 698 (γ
=CH).−
1H-NMR (CDCl3, 200 MHz): δ = 1.44 (s, 6H, 2xCH3), 1.96-2.08 (o, 1H, 3a-H), 2.26-2.40 (o,
1H, 3b-H), 3.62 (dd, 1H, 5a-H), 3.77 (dd, 1H, 5a-H), 4.08 (t, J= 6 Hz, 1H, 2-H), 4.15 (s, 2H,
OCH2), 4.60 (m, 1H, 4-H), 4.56 (s, 2H, OCH2), 4.67 (m, 2H, 2´-H), 5.12 (d, J = 4 Hz, 1H, 1-
H), 7.23-7.53 (m, 12H, ArH), 7.78-7.99 (m, 2H, ArH).–
13C-NMR (CDCl3, 200 MHz): δ = 28.8 (2xCH3), 34.3 (CH2, C-3), 68.0 (C-3´), 72.2 (CH2, C-
5), 73.4 (OCH2), 73.8 (OCH2), 78.3 (CH, C-2), 79.5 (CH2, C-2´), 84.6 (CH, C-4), 85.9 (CH,
C-1), 125.9 (ArC), 127.8-129.1 (ArC), 138.4 (ArC), 138.7 (ArC), 142.0 (ArC), 162.5 (C-1´).–
Anal. Calcd. for C30H33NO4: (471.59): C: 76.41, H: 7.05;
Found: C: 76.34, H: 7.08.−
3-(3-Deoxy-2,5-dibenzyl-
β
-D-ribofuranosyl)benzoic acid (2-30)
O
OBn
BnO
COOH
A solution of oxazoline 2-29 (700 mg, 1.48 mmol) in nitromethane (20 mL) was treated with
methyl iodide (1 mL) and refluxed for 22 h. The solvents were evaporated under reduced
pressure, and the residue was dissolved in methanol (15 mL) and 20 % aqueous KOH (25 mL)
and refluxed for 44 h. Half of the solvent was removed under reduced pressure, and the
mixture was neutralized by the addition of aqueous 1N HCl solution and extracted three times
with ethyl acetate. The organic phase was dried over Na2SO4 and evaporated under reduced
pressure to afford the crude acid 2-30 (450 mg, 72 %) that could be used for the next reaction
without purification. [α]d23 = −32.26 ° (in CH2Cl2) .−
IR (film): 3040 (acid), 2880, 2920, 1690 (C=O), 1454 (ν C=C), 1272 (C-O(C)), 1106 (νas C-
O-C), 698 (γ =CH).−
Experimental Part
88
1H-NMR (CDCl3, 200 MHz): δ = 1.66-1.86 (o, 1H, 3a-H), 2.09-2.22 (o, 1H, 3b-H), 3.51 (dd,
1H, 5a-H), 3.66 (dd, 1H, 5a-H), 3.91 (m, 1H, 4-H), 4.41 (s, 2H, OCH2), 4.60 (s, 2H, OCH2),
4.67 (t, J= 6 Hz, 1H, 2-H), 5.03 (d, J = 4 Hz, 1H, 1-H), 7.26-7.34 (m, 12H, ArH), 7.96-8.10
(m, 2H, ArH).–
13C-NMR (CDCl3, 200 MHz): δ = 34.0 (CH2, C-3), 72.0 (CH2, C-5), 73.3 (OCH2), 73.7
(OCH2), 78.2 (CH, C-2), 84.6 (CH, C-4), 85.9 (CH, C-1), 117.8 (ArC), 127.6-129.8 (ArC),
138.3 (ArC), 138.6 (ArC).–
Anal. Calcd. for C26H26O5: (418.48) C: 74.62, H: 6.26;
Found: C: 74.56, H: 6.24.−
3-(3-Deoxy-2,5-dibenzyl-
β
-D-ribofuranosyl)benzamide (2-31)
O
OBn
BnO
CONH2
A mixture of acid 2-30 (450 mg, 1.12 mmol) thionyl chloride (5 mL) and 2 drops of DMF
were heated to 100 °C for 2 h. The reaction mixture was cooled down, toluene was added and
the solvent was evaporated. The residue was dissolved in dry dichloromethane, cooled to 0 °C
and poured into aqueous ice cold ammonia solution (30 mL). The organic phase was
evaporated, dissolved in ethyl acetate and extracted with water. The ethyl acetate phase was
dried over MgSO4, evaporated and purified by column chromatography (first using CH2Cl2,
then CH2Cl2: MeOH = 18:1) to yield amide 2-31 (400 mg, 88 %), [α]d23 = − 42.73 ° (in
CH2Cl2).–
IR (film): 3355, 3216 (NH2), 2923, 2856, 1720 (C=O), 1697 (amid II), 1452 (ν C=C), 1272
(C-O(C)), 1108 (νas C-O-C), 698 (γ =CH).−
Experimental Part
89
1H-NMR (CDCl3, 200 MHz): δ = 1.92-2.11 (o, 1H, 3a-H), 2.25-2.45 (o, 1H, 3b-H), 3.62 (dd,
1H, 5a-H), 3.76 (dd, 1H, 5a-H), 4.07 (m, 1H, 4-H), 4.55 (s, 2H, OCH2), 4.60 (t, J= 6 Hz, 1H,
2-H), 4.66 (s, 2H, OCH2), 5.12 (d, J = 5 Hz, 1H, 1-H), 6.47 (s, 2H, NH2), 7.23-7.55 (m, 12H,
ArH), 7.45-7.91 (m, 2H, ArH).–
13C-NMR (CDCl3, 200 MHz): δ = 34.3 (CH2, C-3), 72.3 (CH2, C-5), 73.5 (OCH2), 73.8
(OCH2), 78.4 (CH, C-2), 84.4 (CH, C-4), 85.9 (CH, C-1), 124.8 (ArC), 127.2-129.8 (ArC),
134.0 (ArC), 138.2 (ArC), 138.6 (ArC), 142.2 (ArC), 170.2 (CONH2).–
Mass calcd.: 417.19;
MS (CI, HR 7500): 417.3 [M+], 392.3, 340.3, 312.3, 221.1, 185.2, 129.1, 91.0, 55.0, 29.0.−
Anal. Calcd. for C26H27NO4: (417.19): C: 74.80, H: 6.52;
Found: C: 74.23, H: 6.48.−
3-(3-Deoxy-
β
-D-Ribofuranosyl)-benzamide (2-32)
O
OH
HO
CONH2
Amid 2-31 (100 mg, 3.82 mmol) was dissolved in abs. tetrahydrofuran (10 mL) and ethanol
(1 mL) under argon atmosphere. 20 mg palladium/charcoal was added, and stirred under H2-
atmosphere for 10 h. The catalyst was filtered off over Celite and the filtrate evaporated to
afford the product (48 mg, 86 %) as a colorless oil. [α]d23 = −12 ° (in methanol).–
IR (film): 3337 (ν NH2), 2921, 1662 (C=O), 1394, 1272, 1103 (ν C-O(H)), 624 (γ OH).−
1H-NMR (MeOD, 200 MHz): δ = 1.83-2.02 (o, 1H, 3a-H), 2.19-2.41 (o, 1H, 3b-H), 3.72 (m,
2H, 5a,b-H), 4.24 (m, 1H, 4-H), 4.38 (t, J= 6 Hz, 1H, 2-H), 4.87 (d, J = 5 Hz, 1H, 1-H), 7.41-
7.61 (m, 2H, ArH), 7.82 (d, 1H, ArH), 7.93 (s, 1H, ArH).–
Experimental Part
90
13C-NMR (MeOD, 200 MHz): δ = 35.6 (CH2, C-3), 64.7 (CH2, C-5), 78.4 (CH, C-4), 79.5
(CH, C-4), 86.6 (CH, C-1), 125.0 (ArC), 126.8 (ArC), 128.6 (ArC), 129.3 (ArC), 134.0
(ArC), 142.3 (ArC), 162.0 (CONH2).–
Mass calcd.: 237.1;
MS (EI, HR 7500): 237.1 [M+], 219.1 [M−H2O], 150.1, 134.1, 105.0, 91.0, 57.0.−
Anal. Calcd. for C12H15NO4: (210.09): C: 60.75, H: 6.37;
Found: C: 60.46, H: 6.11.−
(2,3,5-Tribenzyl-β-D-ribofuranosyl)-benzene (2-34)
O
OBn
BnO
BnO
A solution of bromo benzene (2-33) (0.194 mL, 1.845 mmol) in anhydrous THF (20 mL) was
treated under nitrogen at −85 °C within 10 min with a solution of n-BuLi (1.23 mL, 1.5 M in
hexane). After 30 min at −85 °C a solution of lactone (2-7) (500 mg,1.23 mmol) in THF (10
mL) was added and stirred for an additional 1 h and then warmed over 2 h to −30 °C (TLC
control). The reaction was then quenched by the addition of water (10 mL) and extracted with
diethyl ether (30 mL). The organic phase was dried with Na2SO4 and evaporated under
reduced pressure to afford a slightly red oil.
The residue was dissolved in CH2Cl2 and treated at −78 °C with boron trifluoride diethyl
etherate (0.63 mL, 2.5 mmol) and triethylsilane (0.375 mL, 2.5 mmol). The reaction mixture
was stirred for 1 h at −78 °C, warmed overnight to 10 °C, neutralized by the addition of a
saturated aqueous sodium hydrogencarbonate solution (ca. 15 mL), and extracted with CH2Cl2
(30 mL). The solution was dried over Na2SO4 and purified by column chromatography
(hexane/ethyl acetate: 6: 1) to afford white crystals (400 mg, 69.5 %), mp.: 55–57 °C, [α]d23 =
−36 ° (in CCl4).–
IR (KBr): 3087, 3064, 3031, 2865, 1454 (ν C=C), 1272 (C-O(C), 1106 (νas C-O-C), 698 (γ
=CH).−
Experimental Part
91
1H-NMR (CDCl3, 200MHz): δ = 3.65 (ddd, Jgem= 10.4 Hz, J4,5a= 4.2 Hz, J4,5b=4.0 Hz; 2H,
5a,b-H), 3.81 (d, J1,2= 6.5 Hz, J2,3= 5.3 Hz, 1H, 2-H), 4.01 (dd, 1H, 3-H), 4.35 (q, 1H, 4-H),
4,47 (dd, AB-system, J= 12 Hz, 2H, OCH2), 4,56 (dd, AB-system, J= 12 Hz, 2H, OCH2), 4,58
(dd, AB-system, J= 12 Hz, 2H, OCH2), 5.02 (d, J1,2= 6.5 Hz, 1H, 1-H), 7.5 (m, 20H, ArH).–
13-NMR (CDCl3, 200MHz): δ = 70.95 (OCH2), 72.44 (OCH2), 72.69 (OCH2), 73.94 (CH2, C-
5), 82.21 (CH, C-2), 83.141 (CH, C-3), 84.27 (CH, C-1), 126.77 (ArC), 128.06−128.82
(ArC), 138.34 (ArC), 138.49 (ArC), 138.68 (ArC), 140.94 (ArC).–
Mass calcd.: 480.23;
MS (EI / 200 °C): m/z (%) = 480.22 (100 %), 389.2 [M+– 91], 181, 105, 57, 23.−
Anal. Calcd. for C32H32O4: (480.59): C: 79.97, H: 6.71;
Found: C: 78.96, H: 6.57.−
3-(2,3,5-Tribenzyl-β-D-ribofuranosyl)-fluoro-benzene (2-37)
O
OBn
BnO
BnO F
A solution of 1-fluoro-3-bromo benzene 2-36 (0.202 mL, 1.845 mmol) in anhydrous THF (20
mL) was treated under nitrogen at −85 °C within 10 min with a solution of n-BuLi (1.23 mL,
1.5 M in hexane). After 30 min at −85 °C a solution of the lactone (2-7) (500 mg, 1.23 mmol)
in THF (10 mL) was added and stirred for an additional 1 h and then warmed over 2 h to −30
°C (TLC control). The reaction was then quenched by the addition of water (10 mL) and
extracted with diethyl ether (30 mL). The organic phase was dried with Na2SO4 and
evaporated under reduced pressure to afford a slightly red oil.
The residue was dissolved in CH2Cl2 and treated at −78 °C with boron trifluoride diethyl
etherate (0.63 mL, 2.5 mmol) and triethylsilane (0.375 mL, 2.5 mmol). The reaction mixture
was stirred for 1 h at −78 °C, let it warm up overnight to 10 °C, neutralized by the addition of
a saturated aqueous sodium hydrogencarbonate solution (ca. 15 mL), and extracted with
Experimental Part
92
CH2Cl2 (30 mL). The solution was dried over Na2SO4 and purified by column
chromatography (hexane/ethyl acetate: 6: 1) to afford 2-37 as a yellow oil (500 mg, 83 %),
[α]d23 = −30.52 ° (in CH2Cl2).–
IR (KBr): 3336, 3031, 2865, 1727, 1614, 1590, 1496, 1454 (ν C=C), 1268 (ν C-F), 1106 (νas
C-O-C), 698 (γ =CH).−
1H-NMR (CDCl3, 200 MHz): δ = 3.76 (t, J4,5a= 4.5 Hz, J4,5b=4.2 Hz, 2H, 5a,b-H), 3.92 (dd,
J1= 5.2 Hz, J2= 0.5 Hz, 1H, 3-H), 4.14 (dd, J1,2= 4.9 Hz, J2,3= 3.9 Hz, 1H, 2-H), 4.48 (q, 1H,
4-H), 4,60 (dd, AB-system, J= 6 Hz, 2H, OCH2), 4,68 (dd, AB-system, J= 3 Hz, 2H, OCH2),
4,72 (s, 2H, OCH2), 5.14 (d, J1,2= 6.8 Hz, 1H, 1-H), 7.43 (m, 20H, ArH).–
13C-NMR (CDCl3, 200 MHz): δ = 70.87 (CH2, C-5), 72.49 (OCH2), 72.88 (OCH2), 74.04
(OCH2), 77.97 (CH, C-4), 82.34 (CH, C-2), 82.45 (CH, C-3), 84.35 (CH, C-1), 113.35 (JC-F=
22.05 Hz, ArC, C-4´), 114.799 (JC-F= 21.05 Hz, ArC, C-2´), 122.43 (JC-F= 2.8 Hz, ArC, C-6´),
128.17-128.91 (ArC, benzyl), 130.17 (JC-F= 8.15 Hz, ArC, C-5´), 138.17, 138.40, 138.55 (q, 3
ArC, benzyl), 143.75 (JC-F= 7.05 Hz, ArC, C-1´), 161.02 (JC-F= 241 Hz, ArC, C-3´).–
Mass calcd.: 498.58;
MS (EI / 200 °C, HR): m/z (%) = 498.22 [M+] (61 %), 407.2 [M+– 91, CH2Ph], 241, 181, 91,
65, 39.−
3-(2,3,5-Tribenzyl-β-D-ribofuranosyl)-anisol (2-40)
O
OBn
BnO
BnO OCH
3
A solution of 3-bromo anisole 2-39 (0.231 mL, 1.845 mmol) in anhydrous THF (20 mL) was
treated under nitrogen at −85 °C within 10 min with a solution of n-BuLi (1.23 mL, 1.5 M in
hexane). After 30 min at −85 °C a solution of the lactone 2-7 (500 mg, 1.23 mmol) in THF
(10 mL) was added and stirred for an additional 1 h and then warmed over 2 h to −30 °C
(TLC control). The reaction was then quenched by the addition of water (10 mL) and
Experimental Part
93
extracted with diethyl ether (30 mL). The organic phase was dried with Na2SO4 and
evaporated under reduced pressure to afford a slightly red oil.
The residue was dissolved in CH2Cl2 and treated at −78 °C with boron trifluoride diethyl
etherate (0.63 mL, 2.5 mmol) and triethylsilane (0.375 mL, 2.5 mmol). The reaction mixture
was stirred for 1 h at −78 °C, let it warm up overnight to 10 °C, neutralized by the addition of
a saturated aqueous sodium hydrogencarbonate solution (ca. 15 mL), and extracted with
CH2Cl2 (30 mL). The solution was dried over Na2SO4 and purified by column
chromatography (hexane/ethyl acetate: 6: 1) to afford 2-40 as a yellow oil (480 mg, 78.2 %),
[α]d23 = −34.4 ° (in CH2Cl2).–
IR (KBr):3345, 3087, 3029, 2911, 2867, 1602, 1454 (ν C=C), 1261 (C-O(C)), 1047 (νas C-O-
C), 696 (γ =CH).−
1H-NMR (CDCl3, 200 MHz): δ = 3.72 (m, 2H, 5a,b-H), 3.78 (s, 3H, OCH3), 3.94 (t, J= 4 Hz,
1H, 3-H), 4.14 (t, J = 4.4 Hz, 1H, 2-H), 4.48 (q, 1H, 4-H), 4,60 (s, 2H, OCH2), 4,67 (d, J= 3
Hz, 2H, OCH2), 4,70 (d, J= 2 Hz, 2H, OCH2), 5.12 (d, J1,2= 6.3 Hz, 1H, 1-H), 6.89 (m, 1H,
ArH), 7.08 (m, 2H, ArH), 7.40 (m, 17H, ArH).–
13C-NMR (CDCl3, 200 MHz): δ = 55.55 (OCH3), 70.88 (CH2, C-5), 72.47 (OCH2), 72.72
(OCH2), 73.97 (OCH2), 78.02 (CH, C-4), 82.14 (CH, C-2), 83.10 (CH, C-3), 84.20 (CH, C-1),
111.84 (ArC, C-4´), 114.12 (ArC, C-2´), 119.11 (ArC, C-6´), 128.07-128.86 (ArC, benzyl),
129.80 (ArC, C-5´), 138.34, 138.47, 138.65 (q, ArC, benzyl), 142.65 (ArC, C-1´), 160.19
(ArC, C-3´).–
Mass calcd.: 510.24;
MS (EI / 200 °C): m/z (%) = 510.24 (61 %), 419.2 [M+–91, CH2Ph], 253.1, 135.1, 91.0,
65.0.−
Anal. Calcd. for C33H34O5: (510.24): C: 77.62, H: 6.71;
Found: C: 77.53, H: 6.98.−
Experimental Part
94
β
-D-Ribofuranosyl-benzene (2-35)
O
OHHO
HO
A solution of benzyl ether 2-34 (100 mg, 0.21 mol) in abs. dichloromethane (10 mL) was
cooled to 0 °C under N2 atmosphere and 1N BBr3 solution (0.65 mL, 0.65 mol) was added
slowly. The reaction was completed in 30 min. (TLC monitoring). The reaction mixture was
neutralized by the addition of sat. aqueous NaHCO3 solution and the water phase was
evaporated under reduced pressure. The residue was dissolved in methanol, filtered, and
purified by column chromatography (CH2Cl2: methanol: 9: 1) to yield 2-35 (39 mg, 88.4 %).
mp.: 120−121 °C, white crystals. [α]d23 = −9.7 ° (in methanol).−
IR (KBr): 3306 (ν OH), 1454 (ν C=C), 1272 (C-O(C)), 1106 (νas C-O-C).−
1H-NMR (CDCl3, 200 MHz): δ = 3.74 (m, 1H, 5a-H), 3.80 (m, 1H, 3-H), 3.83 (m, 1H, 5b-H),
3.90 (m, 1H, 4-H), 4.05 (m, 1H, 3-H), 5.03 (d, J= 6.5 Hz, 1H, 1-H), 7.27−7.54 (m, 5H,
ArH).–
13C-NMR (CDCl3, 200 MHz): δ = 63.2 (CH2, C-5), 79.0 (CH, C-3), 79.2 (CH, C-2), 84.8
(CH, C-4), 87.9 (CH, C-1), 126.4 (ArC), 127.7 (ArC), 127.9 (ArC), 128.5 (ArC), 141.2 (q,
ArC).–
Mass calcd: 210.09;
MS (HR, EI / 200 °C): m/z (%) = 210 (2 %) [M+], 192 (93 %) [M+−H2O], 179, 174, 131, 107,
91, 73.–
Experimental Part
95
β
-D-Ribofuranosyl-3´-fluoro-benzene (2-38)
O
OHHO
HO
F
A solution of benzyl ether 2-37 (110 mg, 0.225 mol) in abs. dichloromethane (10 mL) was
cooled to 0 °C under N2 atmosphere and 1N BBr3 solution (0.7 mL, 0.7 mol) was added
slowly. The reaction was completed in 30 min. (TLC monitoring). The reaction mixture was
neutralized by the addition of sat. aqueous NaHCO3 solution and the water phase was
evaporated under reduced pressure. The residue was dissolved in methanol, filtered, and
purified by column chromatography (CH2Cl2: methanol: 9: 1) to afford 2-38 as white crystals
(38 mg, 74.5 %). mp.: 68 °C, [α]d23 = −6.2 ° (in methanol).−
IR (KBr): 3473, 3413 (ν OH), 1454 (ν C=C), 1272 (C-O(C)), 1256 (ν C-F), 1106 (νas C-O-
C).−
1H-NMR (CDCl3, 200 MHz): δ = 3.68 (m, 1H, 5a-H), 3.79 (m, 1H, 3-H), 3.86 (m, 1H, 5b-H),
4.02 (m, 1H, 4-H), 4.05 (m, 1H, 3-H), 5.11 (d, J= 6.5 Hz, 1H, 1-H), 7.23−7.43 (m, 4H,
ArH).–
13C-NMR (CDCl3, 200 MHz): δ = 62.5 (CH2, C-5), 71.9 (CH, C-3), 78.2 (CH, C-2), 83.6
(CH, C-4), 85.5 (CH, C-1), 112.6 (JC-F= 22.3 Hz, C-4´), 114.0 (JC-F= 21.3 Hz, C-2´), 122.0
(JC-F= 2.8 Hz, C-6´), 129.9 (JC-F= 3.25 Hz, C-5´), 144.0 (JC-F= 7.15 Hz, C-1´), 160.9 (JC-F=
242.65 Hz, C-3´).–
Mass calcd.: 228.08;
MS (EI / 200 °C, HR 7500): m/z (%) = 228.08 (3 %) [M+], 210.1 [M+−F], 192.1 [−H2O],
167.1, 149.0, 125.1, 109.1, 57.0.−
Experimental Part
96
3-
β
-D-Ribofuranosyl-anisol (2-41)
O
OHHO
HO
OCH
3
A solution of benzyl ether 2-40 (250 mg, 0.55 mol) in abs. dichloromethane (10 mL) was
cooled to −78 °C under N2 atmosphere and 1N BBr3 solution (0.7 mL, 0.7 mol) was added
slowly. The reaction was completed in 90 min. (TLC monitoring). The reaction mixture was
neutralized by the addition of sat. aqueous NaHCO3 solution and the water phase was
evaporated under reduced pressure. The residue was dissolved in methanol, filtered, and
purified by column chromatography (CH2Cl2: methanol: 9: 1) to yield 2-41 (101 mg, 85 %).
mp.: 52.5 °C, white crystals. [α]d23 = −7.5 ° (in methanol).−
IR (KBr): 3473, 3413 (ν OH), 1617, 1454 (ν C=C), 1263 (C-O(C)), 1108 (νas C-O-C).−
1H-NMR (CDCl3, 200 MHz): δ = 3.68 (m, 1H, 5a-H), 3.79 (m, 1H, 3-H), 3.86 (m, 1H, 5b-H),
4.02 (m, 1H, 4-H), 4.05 (m, 1H, 3-H), 5.11 (d, J= 6.5 Hz, 1H, 1-H), 7.23−7.43 (m, 4H,
ArH).–
13C-NMR (CDCl3, 200 MHz): δ = 54.9 (OCH3), 63.2 (5-C, CH2), 78.8 (3-C, CH), 84.8 (4-C,
CH) 86.5 (2-C, CH), 87.7 (1-C, CH), 111.75 (4´-C), 113.1 (2´-C), 118.6 (6´-C), 129.5 (5´-C),
143.0 (1´-C), 160.2 (3´-C).–
Mass calc.: 240.1;
MS (EI / 200 °C, HR 7500): m/z (%) = 240.1 (18 %) [M+], 222.2 [M+−H2O], 183.2, 152.1,
137.1, 109.1, 97.1, 57.1.−
(S)-(+)-
γ
-Carbonyl-butyrolactone (2-43)[37]
O
HOOC
H
O
Experimental Part
97
A solution of NaNO2 (12,6 g, 0.183 mol) in H2O (27 mL) was added dropwise to the solution
of L-glutamic acid (2-42) (18 g, 0.122 mol) in water (48 mL)and cc. HCl (25.2 mL)at 0 °C
and stirred for 6 h. The colorless reaction mixture was warmed up to room temperature
overnight. Evaporation of water under reduced pressure resulted in a mixture of white crystals
and a colorless oil. This residue was dissolved in ethyl acetate and filtered. The organic phase
was dried over NaSO4 and the solvent evaporated. The product was isolated as a viscous oil
14.64 g (92%). Recrystallisation from ethyl acetate/hexane yielded white powder. mp.: 71-73
°C; [α]d20 = +15.6 ° (c = 2.0 in EtOH).−
IR (KBr): 1775 (lacton C=O), 1723 (acid C=O), 1175 (C–O).–
1H-NMR (CD3OD, 200 MHz): δ = 1.8-2.3 (m, 4H, CH2–CH2), 4.2 (m, 1H, CH-O).–
13C-NMR (DMSO-d6 200 MHz): δ = 26.0 (CH2), 27.1 (CH2), 76.0 (CH), 172.5 (C=O), 177.5
(C=O).–
(S)-(+)-
γ
-Ethoxycarbonyl-
γ
-butyrolactone (2-44a), diethyl-
α
-hydroxyglutarate (2-44b)[37]
O
EtOOC O
+
HO COOEt
HEtOOC
A solution of (S)-(+)-
γ
-carbonyl-butyrolactone 2-43 (14.0 g, 0.107 mol) and p-TsOH (0.6 g,
3.8 mmol) in ethanol (30 mL) and benzene (70 mL) was heated under reflux for 7.5 h. The
solvents were distilled off. The residue was dissolved in benzene and extracted with H2O,
aqueous 10 % Na2CO3, and H2O. The organic phase was dried over NaSO4 and evaporated. A
colorless oil was obtained, which was used in the next step without purification.−
2,3-Didesoxy-
γ
-ribonolacton (2-45)[37]
O
HO O
To a suspension of NaBH4 (2.0 g, 0.052 mol) in ethanol (30 mL) the mixture of (S)-(+)-
γ
-
ethoxycarbonyl-
γ
-butyrolacton (2-44a) and diethyl-
α
-hydroxyglutarat (2-44b) (12.64 g) in
Experimental Part
98
ethanol (40 mL) was added slowly at room temperature. After 90 min. the pH value of the
solution was set to pH= 3 with aqueous 10 % HCl solution at 0 °C. The precipitate was
filtered off and the filtrate was evaporated under reduced pressure. The residue was purified
by column chromatography (dichloromethane: methanol : 9:1) to afford 2-45 as a colorless oil
(8.2 g, 71 %). [α]d20 = +29.6 ° (c = 0.4 in EtOH).−
IR (KBr): 3400 (O-H), 1765 (lacton C=O), 1180 (C–O).–
1H-NMR (CDCl3, 200 MHz): δ = 2.0-2.8 (m, 4H, CH2-CH2), 3.55-4.06 (m, 2H, 5a,b-H), 4.66
(m, 1H, CH).–
Anal. Calcd. for C5H8O3: (116.05): C 51.72, H 6.94;
Found: C 51.67, H 6.95.−
5-Benzyl-2,3-didesoxy-
γ
-ribonolacton (2-46)[38]
O
BnO O
Freshly prepared Ag2O (4.05 g, 0.017 mol) was suspended to a solution of 2,3-didesoxy-
γ
-
ribonolactone (2-45) (1.35 g, 0.011 mol) in abs. DMF (20 mL). Benzyl bromide (2.07 mL)
was added. The reaction mixture was protected from light and stirred at room temperature for
2 days. The unsolved material was filtered off and washed with CHCl3 (50 mL). The filtrated
was kept in the refrigerator overnight and pyridine (5 mL) was added. The solution was
washed with H2O, aqueous 10 % HCl, H2O, aqueous 10 % Na2CO3 solution, and H2O
consecutively. The organic phase was dried over Na2SO4. After evaporation of the solvents
the residue was purified by column chromatography (dichloromethane: methanol: 9:1) to
afford 2-46 (1.3 g, 60 %) as a yellow oil. F.p.Lit 160-164°/0.02 mm[37], [α]d20 = +18.1 ° (c =
2.70, EtOH).−
IR (KBr): 3064, 2925, 1789 (C=O), 1454 (ν C=C), 1272 (C-O(C)), 1122 (C-O(C)), 698 (
γ
=CH).−
Experimental Part
99
1H-NMR (CDCl3, 200 MHz): δ = 2.38 (m, 2H, 3-H), 2.48 (m, 2H, 2-H), 3.41-3.66 (m, 2H,
5a,b-H), 4.43 (s, 2H, OCH2), 4.70 (m, 1H, 4-H), 7.29 (m, 5H, ArH).−
13C-NMR (CDCl3, 200 MHz): δ = 26.01 (CH2, C-3), 27.48 (CH2, C-2), 71.68 (CH2, C-5),
73.19 (OCH2), 73.81 (CH, 4-C), 127.83 (ArC), 128.04 (ArC), 128.26 (ArC), 128.36 (ArC),
137.42 (q, ArC), 176.87 (C=O). −
2-Deoxy-ribonolactone (2-48)[117]
O
HO
O
HO
A solution of 2-deoxy-D-ribose 2-47 (3.0 g, 11.36 mmol) and water (100 mL) was cooled to
0 °C. Bromine (6 mL, 0.108 mol) was added dropwise. The solution was kept at 0 °C for 30
minutes and stirred at room temperature for 5 days.
The reaction mixture was extracted with diethyl ether. Evaporation of the water phase resulted
in a pale yellow viscous oil (3 g), which was used for the next step without further
purification.
3,5-Dibenzyl-2-deoxy-ribonolactone (2-52)[118]
O
BnO
O
BnO
The mixture of 2-deoxy-ribonolactone (2-48) (1.5 g, 11.36 mmol) and benzyl trichloro-
acetimidate (2-51) (12.57 g, 47.77 mmol) was dissolved in dry dioxan (25 mL) and trifluoro-
methansulfonic acid (0.3 mL) was added. The reaction mixture was stirred at room
temperature overnight. The solvent was distilled off at reduced pressure. The residue was
dissolved in dichloromethane and extracted with water. The organic phase was dried over
MgSO4, and evaporated to obtain a yellow oil, and white crystals. Byproduct trichloro-
acetamide was crystallized from pentane: dichloromethane 1:1, and the residue was purified
by column chromatography (PE: EE:3: 1) to afford pure 2-52 (3.1g, 87.4 %) as a yellow oil.
[α]d20 = +31° (c = 2.0, CHCl3)[118].−
Experimental Part
100
IR: 3064, 2925, 1789 (C=O), 1454 (ν C=C), 1272 (C-O(C)), 1122 (C-O(C)), 698 (γ =CH).−
1H-NMR (200 MHz): δ = 2.64 (dd, J3,2a = 2.4 Hz, Jgem = 18.1 Hz, 1H, 2a-H), 2.87 (dd, J3,2b =
4.2 Hz, Jgem = 18.1 Hz, 1H, 2b-H), 3.68 (m, 2H, 5a,b-H), 4.30 (dt, J3,2a = 2.1 Hz, J3,2b = 6 Hz,
1H, 3-H), 4,56 (2s, 4H, OCH2), 4.65 (m, 1H, 4-H), 7.32−7.40 (m, 10H, ArH).−
13C-NMR (200 MHz): δ = 36.1 (CH2, C-2), 70.0 (CH2, C-5), 71.6 (OCH2), 74.1 (OCH2), 76.5
(CH, C-4), 84.5 (CH, C-3), 128.0-129.3 (ArC), 137.4 (q, ArC), 137.7 (q, ArC), 176.1 (C=O).−
8.3 Experimental Part to Chapter 3
3-(2,3-Isopropyliden-
β
-D-ribofuranosyl)benzamide (3-2)
O
OO
HO
CONH2
A solution of the amide (2-2) (300 mg, 1.2 mmol) in anhydrous acetone (40 mL) was treated
with concentrated sulfuric acid (1 mL) at 0 °C and stirred for 18 h at 20 °C. The solution was
then neutralized by the addition of a saturated aqueous solution of sodium hydrogen carbonate
and evaporated under reduced pressure. The residue was dissolved in ethyl acetate, filtered,
and evaporated under reduced pressure. The product was purified by column chromatography
(CH2Cl2 / 5 % MeOH) to afford the acetonide (320 mg, 93 %) as a white foam: [α]d23 = −32 °
(c = 0.1, MeOH).–
IR (CCl4): 3531, 3415, 3010, 2996, 2938, 1677, 1587, 1384, 1375.–
UV: 207 (3.24), 226 (sh, 2.96), 272 (1.87), 280 (1.78).–
Experimental Part
101
1H-NMR (300 MHz): δ = 1.31 (s, 1H, CH3), 1.59 (s, 1H, CH3), 3.73 (dd, J4,5a = 4.0 Hz, Jgem =
12.3 Hz, 1H, 5a-H), 3.89 (dd, J4,5b = 2.6 Hz, Jgem = 12.3 Hz, 1H, 5b-H), 4.14 (m, 1H, 4-H),
4.26 (s, 1H, OH), 4.45 (dd, J2,3 = 6.8 Hz, J1,2 = 5.5 Hz, 1H, 2-H), 4.72 (dd, J2,3 = 6.8 Hz, J3,4 =
4.2 Hz, 1H, 3-H), 4.84 (d, J1,2 = 5.5 Hz, 1H, 1-H), 6.64-7.15 (s, 2H, NH2), 7.33 (t, J = 7.7 Hz,
1H, 5´-H), 7.45 (d, J5,6 = 7.7 Hz, 1H, 4´-H), 7.70 (d, J4,5 = 7.7 Hz, 1H, 6´-H), 7.92 (s, 1H, 2´-
H).–
13C-NMR (300 MHz): δ = 24.98 (CH3), 27.02 (CH3), 61.74 (CH2, C-5), 80.78 (CH), 84.85
(CH), 86.26 (CH), 114.59 (q, (CH3)2C), 124.06 (ArC), 126.60 (ArC), 128.21 (ArC), 129.21
(ArC), 133.02 (q, ArC), 139.68 (q, ArC), 169.72 (q, C=O).–
Anal. Calcd. for C15H19O5: (293.13): C 61.42, H 6.53, N 4.78;
Found: C 61.40, H 6.57, N 4.70.−
3-(
β
-D-Ribofuranosyl)benzamide-5´-phosphate bisodium salt (3-3)
O
O
HO OH
CONH
2
PNaO
ONa
O
A solution of the acetonide (3-2) (1.0 g, 3.4 mmol) and freshly distilled phosphoryl chloride
(0.75 mL, 8.1 mmol) in trimethyl phosphate (7.0 mL, 60.0 mmol) was stirred for 4 h at 0 °C
and 6 h at 5 °C. The reaction was hydrolyzed with ice-water (16.0 g) and extracted with
diethyl ether (30 mL). (TLC: on Cellulose F, isopropanol/water/ammonia = 7:2:1).
The aqueous phase was adjusted to pH 1.5 by the addition of 1 N NaOH and heated for 45
min at 70 °C. The solution was then neutralized with 1 N NaOH and evaporated under
reduced pressure. The residue was purified by column chromatography (DEAE-sephadex;
gradient: 1 L of water/l L of 0.18 M TEAB-buffer, pH 7.5) to afford the pseudonucleotide
(778 mg, 53 %) in form of the TEAE-salt. The conversion to the disodium salt was effected
with ion exchange resin; [α]23 −24.4 ° (c = 0.1, MeOH).–
IR (KBr): 3340 (OH), 2940 (C-H), 1680 (C=O), 1617, 1450, 1230 (phosphate), 1190, 1100
(ether), 1050 (phosphate), 918, 856.–
Experimental Part
102
1H-NMR (D2O, 300 MHz): δ = 3.65 (t, 1H, 3-H), 3.91 (q, 1H, 4-H), 4.01 (dd, J2,3 = 5.4 Hz,
J1,2 = 7.1 Hz, 1H, 2-H), 4.03, 4.28 (2 dd, J4,5 = 4.4 Hz, Jgem = 11.7 Hz, 2H, 5-H) , 5.11 (d, J1,2
= 7.1 Hz, 1H, 1-H), 7.41 (t, J = 7.7 Hz, 1H, 5´-H), 7.56 (d, J5,6 = 7.7 Hz, 1H, 4´-H), 7.77 (d,
J4,5 = 7.7 Hz, 1H, 6´-H), 7.96 (s, 1H, 2´-H).–
13C-NMR (D2O, 300 MHz): δ = 65.13 (C-5), 72.33 (CH), 77.64 (CH), 83.83 (CH), 84.41
(CH), 126.01 (ArC), 128.25 (ArC), 129.80 (ArC), 131.36 (ArC), 133.46 (ArC), 140.11 (q,
ArC), 173.01 (q, C=O).–
31P-NMR (D2O, 300 MHz): δ = 3.71.−
Anal. Calcd. for C12H14NNa2O8P: (377.03): C: 38.21, H: 3.74, N: 3.71;
Found: C: 34.17, H: 4.64, N: 3.67.−
3-(
β
-D-Ribofuranosyl)benzamide-adenine-dinucleotide (3-5)
O
OHHO
O
CONH2
POPO
OO
OH OH
O
OHHO
N
N
N
N
NH2
The corresponding free acid monophosphate 3-3 (0.1 mmol) was rigorously dried and
suspended in DMF (0.5 mL). Carbonyldiimidazole (0.5 mmol) was added and the reaction
mixture became homogeneous in a few minutes. After 3 h, HPLC analysis showed the
disappearance of starting material tR= 13−15 min) and the appearance of a peak at tR= 4−5
min. Methanol (33 pL) was added to hydrolyze excess carbonyldiimidazole and after 30 min,
AMP (adenosine monophosphate) (0.15 mmol), dissolved in of anhydrous DMF (2 mL)
containing tri-n-butylamine (0.15 mmol), was added. The reaction mixture was stirred for 48
h at room temperature and monitored by HPLC. When the reaction was completed, 5 mL of
water was added and the solution was reduced to dryness. The gummy residue was dissolved
again in 20 mL of water containing sodium acetate (30 mg) and extracted twice with 20 mL
Experimental Part
103
each of chloroform and diethyl ether. The aqueous layer was treated with triethylamine (200
pL) until pH 10 was reached and stirred for 8−24 h. After such time, the entire mixture was
lyophilized and the residue chromatographied on a Hamilton HA-X4 anion-exchange column
(HCO2-form, 0.8 x 28 cm). During a 7 h gradient from water to 2 M (H4N)HCO2, with a flow
rate of 2 mL/min, samples of 12 mL were collected and monitored by W at 290 nm. The
desired fractions were collected and passed through a strong cation-exchange resin (AG50W-
X8, H+ form, 1.5 x 15 cm) and eluted with water. After collection of 15 mL fractions from
this column, the product was always present in fractions 2-4. These fractions were lyophilized
to give the dinucleotide as white fluffy solids. Yield 71 %.
1H-NMR (D2O, 300 MHz): δ = 4.07 (dd, 1H, 2´(B)-H, J1´,2´= 7.2 Hz, J2´,3´= 5.3 Hz), 4.15-4.26
(m, 6H, 3´(B)-H, 4´(B)-H, 5´,5´´(A)-H, 5´,5´´(B)-H), 4.35 (m, 1H, 4´(A)-H), 4.44 (pseudo t,
1H, 3´(A)-H), 4.59 (pseudo t, 1H, 2´(A)-H), 4.75 (d, 1H, 1´-(B)-H, J1´,2´= 7.2 Hz), 6.01 (d,
1H, 1´(A)-H, J1´,2´= 5.3 Hz), 7.41 (t, 1H, 5-H, J= 7.7 Hz), 7.57 (d, 1H, 4-H, J= 7.7 Hz), 7.66
(d, 1H, 6-H), 7.73 (s, 1H 2(B)-H], 8.31 (s, 1H, 2-H), 8.55 (s, 1H, H8-(A)-H).−
31P-NMR (D2O), AB-system: δ = 9.36 P1, 9.59 P2, JP,P= 21.2 Hz .−
8.4 Experimental part to Chapter 4.
Condensation of amides with acid chlorides, General Procedure A and B.
Procedure A: To a boiling solution of the amide (10 mmol) in dry toluene (20 mL) a solution
of the acid chloride (5 mmol) in toluene (5 mL) was added dropwise. The mixture was
refluxed overnight (TLC monitoring), the solvent was removed under reduced pressure and
the residue was purified by column chromatography on silica gel (dichloromethane) and
recrystallized from diethyl ether. (For yields and mp of 4-5a to 4-5h see Table 4-1.)
Procedure B: Alternatively, the sodium salt of the amide (prepared by reaction of the amide
solution with sodium hydride) was employed, with fumaric acid monoethyl ester anhydride.
Experimental Part
104
4-Benzoylamino-4-oxobut-2-enoic acid ethyl ester (4-5a)
NOCH
2
CH
3
O O
OH
Benzamide (4-2) (1.23 g, 10 mmol) in dry toluene (20 mL) was reacted according to
procedure A with fumaric acid monoethyl ester chloride (4-3a) (1.78 g, 7.5 mmol) in toluene
(5 mL) to yield 4-5a (1.18 g, 51 %), mp: 87–89 °C.−
IR (KBr)
ν
~
= 3291 cm-1 (N-H), 1724 (C=O), 1703 (C=O), 1672 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 1.36 (t, J = 7 Hz, 3H, CH3), 4.32 (q, J = 7 Hz, 2H, CH2),
6.90 (d, J = 2 Hz, 2H, CH), 7.02 (d, J = 2 Hz, 2H, CH), 7.61 (m, 3H, ArH), 7.96 (m, 2H,
5ArH), 9.43 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 14.6 (CH3), 61.8 (CH2), 128.5 (CH), 129.4 (CH), 133.3
(ArC), 134.1 (ArC), 134.3 (ArC), 135.2 (ArC), 165.4 (C=O), 166.3 (C=O), 167.3 (C=O).−
Anal. Calcd for C12H11NO4: (233.22): C 63.09, H 5.26;
Found: C 61.66, H: 5.29.−
4-(2-Fluorobenzoylamino)-4-oxobut-2-enoic acid ethyl ester (4-5b)
NOCH
2
CH
3
O O
OH
F
2-Fluorobenzoic acid amide (2.0 g, 14.4 mmol) in dry toluene (15 mL) was reacted with
fumaric acid chloride (4-3a) (1.3 g, 9.6 mmol) as described in procedure A to afford imide 4-
5b (890 mg, 42 %), mp: 54−55 °C.−
IR (KBr)
ν
~ = 3388 cm-1 (N-H), 1720 (C=O), 1705 (C=O), 1678 (C=O).−
Experimental Part
105
1H-NMR (200 MHz, CDCl3): δ = 1.35 (t, J = 7 Hz, 3H, CH3), 4.30 (q, J = 7 Hz, 2H, CH2),
6.94 (d, J = 14 Hz, 1H, CH), 7.38 (m, 2H, ArH), 7.61 (m, 1H, ArH), 7.88 (d, J = 14 Hz, 1H,
CH), 8.08 (m, 1H, ArH), 9.19 (d, J = 13, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 14.5 (CH3), 61.8 (CH2), 116.9 (CH), 120.4 (ArC), 125.8
(CH), 132.7 (ArC), 134.7 (ArC), 135.4 (ArC), 135.8 (ArC), 158.5 (C=O), 162.5 (C=O), 163.5
(C=O), 165.6 (ArC).−
Anal. Calcd for C13H12FNO4: (265.24): C 58.87, H 4.56, N 5.28;
Found: C 58.39, H 4.46, N 5.24.−
4-(4-Nitrobenzoylamino)-4-oxobut-2-enoic acid ethyl ester (4-5c)
NOCH
2
CH
3
O O
OH
O
2
N
4-Nitrobenzoic acid amide (550 mg, 3.3 mmol) was reacted with fumaric acid chloride (4-3a)
(200 mg, 1.5 mmol) as described in procedure A to afford imide 4-5c (158 mg, 44 %) , mp:
135–137 °C.−
IR (KBr)
ν
~ = 3244 cm-1 (N-H), 1720 (C=O), 1713 (C=O), 1678 (C=O).−
1H-NMR (200 MHz, DMSO): δ = 1.28 (t, J = 7 Hz, 3H, CH3), 4.24 (q, J = 7 Hz, 2H, CH2),
6.75 (d, J = 16 Hz, 1H, CH), 7.54 (d, J = 16 Hz, 1H, CH), 8.15 (d, J = 9 Hz, 2H, ArH), 8.37
(d, J = 9 Hz, 2H, ArH), 11.73 (s, 1H, NH).−
13C-NMR (200 MHz, DMSO): δ = 14.8 (CH3), 59.6 (CH2), 134.4 (CH), 137.8 (CH), 123.7
(ArC), 123.8 (ArC), 128.2 (ArC), 128.3 (ArC), 139.4 (ArC), 150.6 (ArC), 165.1 (C=O), 165.5
(C=O), 166.4 (C=O).–
Anal. Calcd for C13H12N2O6 (292.24): C 53.43, H 4.14, N 9.59;
Found: C: 55.29, H 4.53, N 8.56.−
Experimental Part
106
4-(2-Methoxybenzoylamino)-4-oxobut-2-enoic acid ethyl ester (4-5d)
NOCH
2
CH
3
O O
OH
OCH
3
2-Methoxybenzoic acid amide (2.1 g, 13.9 mmol) was reacted with fumaric acid chloride (4-
3a) (1.0 g, 7.4 mmol) as described in procedure A to afford imide 4-5d (785 mg, 46 %), mp:
98–100 °C.−
IR (KBr)
ν
~
= 3303 cm-1 (N-H), 1720 (C=O), 1705 (C=O), 1684 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 1.38 (t, J = 7 Hz, 3H, CH3), 4.09 (s, 3H, OCH3), 4.32 (q, J =
7 Hz, 2H, CH2), 6.94 (d, J = 14 Hz, 1H, CH), 7.18 (m, 2H, ArH), 7.62 (m, 1H, ArH), 8.00 (d,
J = 14 Hz, 1H, CH), 8.24 (m, 1H, ArH), 10.44 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 14.5 (CH3), 56.7 (OCH3), 61.7 (CH2), 112.2 (CH), 120.1
(ArC), 122.2 (CH), 133.3 (ArC), 135.6 (ArC), 136.3 (ArC), 136.9 (ArC), 158.2 (ArC), 164.2
(C=O), 165.6 (C=O), 166.4 (C=O).−
Anal. Calcd for C14H15NO5 (277.27): C 60.64, H 5.45, N 5.05;
Found: C 60.38, H 5.47, N 5.19.−
4-(4-Methoxybenzoylamino)-4-oxobut-2-enoic acid ethyl ester (4-5e)
NOCH
2
CH
3
O O
OH
H
3
CO
4-Methoxybenzoic acid amide (500 mg, 3.3 mmol) in dry toluene (8 mL) was reacted with
fumaric acid chloride (4-3) (240 mg, 1.8 mmol) as described in A to afford 4-5e (185 mg,
45 %), mp: 120–122 °C.−
IR (KBr)
ν
~
= 3271 cm-1 (N-H), 1726 (C=O), 1709 (C=O), 1668 (νC=O).−
Experimental Part
107
1H-NMR (200 MHz, CDCl3): δ = 1.38 (t, J = 7 Hz, 3H, CH3), 3.93 (s, 3H, OCH3), 4.32 (q, J =
7 Hz, 2H, CH2), 6.98 (d, J = 14 Hz, 1H, CH), 7.03 (d, J = 8 Hz, 2H, ArH), 7.89 (d, J = 8 Hz,
2H, ArH), 8.04 (d, J = 14 Hz, 1H, CH), 8.70 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 14.5 (CH3), 55.9 (OCH3), 61.7 (CH2), 114.6 (CH), 124.6
(ArC), 130.8 (CH), 133.8 (ArC), 135.4 (ArC), 136.7 (ArC), 137.5 (ArC), 164.4 (ArC), 165.5
(C=O), 165.7 (C=O), 167.5 (C=O).−
Anal. Calcd for C14H15NO5 (277.27): C 60.64, H 5.45, N 5.05;
Found: C: 59.97, H: 5.62, N 5.22.−
4-(3,5-Dimethoxybenzoylamino)-4-oxobut-2-enoic acid ethyl ester (4-5f)
NOCH2CH3
O O
OH
H3CO
OCH3
3,4-Dimethoxy-benzoic acid amide (500 mg, 2.8 mmol) in dry toluene (8 mL) was reacted
with fumaric acid chloride (4-3a) (200 mg, 1.5 mmol) as described in procedure A to afford
4-5f (362 mg, 43 %), mp: 162–164 °C.−
IR (KBr)
ν
~
= 3271 cm-1 (N-H), 1722 (C=O), 1705 (C=O), 1675 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 1.38 (t, J = 7 Hz, 3H, CH3), 3.88 (s, 6H, 2 x OCH3), 4.33 (q,
J = 7 Hz, 2H, CH2), 6.72 (d, J = 14 Hz, 1H, CH), 7.01 (s, 1H, ArH), 7.30 (d, J = 14 Hz, 1H,
CH), 8.02 (d, J = 8 Hz, 2H, ArH), 8.78 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 14.5 (CH3), 56.1 (OCH3), 61.6 (OCH2), 112.6 (CH), 124.8
(ArC), 130.4 (CH), 133.2 (ArC), 136.1 (ArC), 136.8 (ArC), 137.9 (ArC), 164.4 (ArC), 165.8
(C=O), 166.1 (C=O), 167.3 (C=O).−
Anal. Calcd for C15H17NO6 (307.30): C 58.63, H 5.58, N 4.56;
Found: C 59.77, H 5.52.−
Experimental Part
108
4-(3,4,5-Trimethoxybenzoylamino)-4-oxobut-2-enoic acid ethyl ester (4-5g)
NOCH2CH3
O O
OH
H3CO
OCH3
H3CO
The sodium salt of the amide was prepared by stirring a mixture 3,4,5-trimethoxy-benzoic
acid amide (4 g, 18.9 mmol) and NaH (1 g, 20.8 mmol) in THF (100 mL) for 30 min, and
reacted with fumaric acid monoethyl ester anhydride (5.1 g, 18.9 mmol) in 30 mL THF
(procedure B). After column chromatography, 4-5g was isolated (4.1 g, 65 %). mp:
114−116 °C.−
IR (KBr)
ν
~ = 3255 cm-1 (N-H), 1729 (C=O), 1707 (C=O), 1672 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 1.38 (t, J = 7 Hz, 3H, CH3), 3.97 (s, 9H, 3 x OCH3), 4.32 (q,
J = 7 Hz, 2H, CH2), 6.94 (d, J = 14 Hz, 1H, CH), 7.21 (s, 2H, ArH), 8.05 (d, J = 14 Hz, 1H,
CH), 9.42 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 14.5 (CH3), 56.6 (OCH3), 56.7 (OCH3), 61.3 (OCH3), 61.9
(OCH2), 106.2 (CH), 124.7 (ArC), 127.1 (CH), 133.6 (ArC), 135.3 (ArC), 143.2 (ArC), 153.3
(ArC), 165.2 (ArC), 166.0 (C=O), 168.5 (C=O), 169.2 (C=O).−
Anal. Calcd for C16H19NO7 (337.32): C 56.97, H 5.68.;
Found: C 56.91, H 6.02.−
4-Benzoylamino-4-oxobut-2-enoic acid octyl ester (4-5h)
NOC
8
H
15
O O
OH
The sodium salt of benzamide was prepared by stirring a mixture of benzamide (500 mg, 4.1
mmol) and NaH (172 mg, 4.1 mmol) in THF (35 mL) for 30 min, and reacted with fumaric
acid monooctyl ester anhydride (1.82 g, 4.1 mmol) in THF (15 mL) (procedure B). After
column chromatography, 4-13 was isolated as an oil (190 mg, 13 %).−
Experimental Part
109
IR (KBr)
ν
~ = 3292 cm-1 (N-H), 1724 (C=O), 1709 (C=O), 1687 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 0.91 (t, J = 6 Hz, 3H, CH3), 1.31 (m, 10H, CH2), 4.25 (q, J
= 3 Hz, 2H, CH2), 6.95 (d, J = 14 Hz, 1H, CH), 7.55 (m, 2H, ArH), 7.68 (m, 1H, ArH), 7.98
(m, 2H, ArH), 8.06 (d, J = 14 Hz, 1H, CH), 9.02 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 14.5 (CH3), 23.1 (CH2), 26.3 (CH2), 28.9 (CH2), 29.6
(CH2), 32.2 (CH2), 66.1 (OCH2), 112.6 (CH), 128.4 (ArC), 129.4 (CH), 132.6 (ArC), 134.0
(ArC), 134.3 (ArC), 135.2 (ArC), 165.6 (ArC), 166.3 (C=O), 166.7 (C=O), 167.0 (C=O).−
Anal. Calcd for C19H25NO4 (331.41): C 68.86, H 7.60;
Found: C 69.67, H 7.51.−
4-Oxo-4-phenylacetylaminobutyric acid methyl ester (4-10)
NOCH
3
O O
OH
Phenylacetic acid amide (4-6a) (1.35 g, 10 mmol) in dry toluene (20 mL) was reacted with
succinic acid monomethyl ester chloride (4-7)[119] (1.65 g, 12.26 mmol) as described in
general procedure A to afford 692 mg (28 %) of 4-10.−
IR (KBr):
ν
~= 3260 cm-1, 1735 (C=O), 1705 (C=O), 1657 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 2.69 (t, J = 2 Hz, 4H, CH2), 3.69 (s, 2H, PhCH2), 3.70 (s,
3H, OCH3), 7.37 (m, 5H, ArH), 9.05 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 28.7 (CH2), 28.9 (CH2), 39.3 (PhCH2), 50.4 (CH3), 127.4
(ArC), 129.0 (ArC), 129.8 (ArC), 135.9 (ArC), 170.7 (C=O), 172.0 (C=O), 175.2 (C=O).−
Anal. Calcd for C13H15NO4: (249.26): C 62.58, H 6.01;
Found: C: 61.33, H: 5.33.−
Experimental Part
110
4-Benzoylamino-4-oxobutyric acid methyl ester (4-11a)
NO
O O
OH
Benzamide (4-2) (1.23 g, 10 mmol) was reacted with succinic acid monomethyl ester chloride
(4-7) (1.65 g, 12.26 mmol) in toluene (5 mL) according to general procedure A to afford 4-
11a (1.06 g, 43 %).−
IR (KBr)
ν
~
= 3291 cm-1 (N-H), 1740 (C=O), 1709 (νC=O), 1677 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 2.72 (t, J = 4 Hz, 2H, CH2), 3.34 (t, J = 4 Hz, 2H, CH2),
3.70 (s, 3H, OCH3), 7.61 (m, 3H, ArH), 7.93 (m, 2H, ArH), 9.35 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 28.6 (CH2), 33.3 (CH2), 52.3 (OCH3), 128.3 (ArC), 129.3
(ArC), 132.9 (ArC), 133.6 (ArC), 166.2 (C=O), 173.4 (C=O), 175.7 (C=O).−
Anal. Calcd for C12H13NO4: (235.24): C 61.22, H 5.53;
Found: C 61.23, H 5.45.−
4-(4-Hydroxybenzoylamino)-4-oxobutyric acid ethyl ester (4-11c)
NO
O O
OH
HO
A solution of 4-benzyloxybenzamide (500 mg, 2.2 mmol) in dry THF (10 mL) was treated
with NaH (88 mg, 2.2 mmol) and stirred for 1 h at 20 °C. Fumaric acid monoethyl ester
anhydride (600 mg, 2.2 mmol) was added and the mixture was heated overnight (procedure
B). The product was purified by column chromatography on silica gel (CH2Cl2) and
crystallized from diethyl ether. A sample of the resulting benzyl ether (150 mg) was dissolved
in dry THF (10 mL) and hydrogenated over palladium/charcoal (10 %) for 3 h. The reaction
mixture was filtered and the solvent removed at reduced pressure. The residue was purified by
column chromatography on silica gel (CH2Cl2) to yield 4-11c (76 mg, 69 %), mp: 146–148
°C.
Experimental Part
111
IR (KBr)
ν
~ = 3091 cm-1 (N-H), 1715 (C=O), 1675 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 1.31 (t, 3H, CH3), 2.76 (t, J = 6 Hz, 2H, CH2), 3.78 (t, J = 6
Hz, 2H, CH2), 4.22 (q, 2H, CH2), 7.76 (d, J = 7 Hz, 2H, ArH), 8.11 (d, J = 7 Hz, 2H, ArH),
8.62 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 14.1 (CH3), 29.2 (CH2), 29.6 (CH2), 61.7 (CH2), 116.5
(ArC), 117.0 (ArC), 125.9 (ArC), 128.8 (ArC), 129.7 (ArC), 161.1 (ArC), 168.2 (C=O), 172.3
(C=O), 172.4 (C=O).−
Anal. Calcd for C13H13NO5 (263.25): C 59.31, H 4.98;
Found: C 59.38, H 4.95.−
3-Oxo-3-phenylacetylaminopropionic acid methyl ester (4-12)
NOCH
3
O O
H
O
Phenylacetic acid amide (4-6a) (1.0 g, 7.4 mmol) was reacted with malonic acid monomethyl
ester chloride (4-8)[120] (1.22 g, 8.1 mmol) in toluene (5 mL) to afford 4-12 (520 mg, 30 %)
after column chromatography (petroleum ether/ethyl acetate 3/1), mp 93−96 °C.−
IR (KBr)
ν
~
= 3266 cm-1 (N-H), 1750 (C=O), 1735 (C=O), 1698.2 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 3.76 (s, 2H, CH2), 3.78 (s, 3H, OCH3), 3.85 (s, 2H, ArCH2),
7.31 (m, 3H, ArH), 7.40 (m, 2H, ArH), 8.62 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 44.2 (CH2), 44.8 (CH2), 53.1 (OCH3), 128.3 (ArC), 129.6
(ArC), 129.9 (ArC), 135.9 (ArC), 170.7 (C=O), 171.8 (C=O).−
Anal. Calcd for C12H13NO4: (235.24): C 61.22, H 5.53;
Found: C 60.85, H 5.28.−
Experimental Part
112
3-Methoxy-N-phenylacetylbenzamide (4-13)
N
O O
H
OCH
3
Phenylacetic acid amide (4-6a) (1.35 g, 10 mmol) in dry toluene (20 mL) was reacted with 3-
methoxybenzoic acid chloride (1.26 g, 9.4 mmol) as described in procedure A to afford imide
4-13 (926 mg, 35 %), mp: 112–114 °C.−
IR (KBr)
ν
~
= 3312 (N-H) cm-1, 1709 (C=O), 1683 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 3.86 (s, 3H, OCH3), 4.36 (s, 2H, PhCH2), 7.17 (t, J = 2 Hz,
1H, ArH), 7.37 (m, 8H, ArH), 9.05 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 44.3 (CH2), 55.9 (OCH3), 113.0 (ArC), 119.9 (ArC), 120.2
(ArC), 127.6 (ArC), 129.0 (ArC), 130.2 (ArC), 130.4 (ArC), 134.1 (ArC), 134.4 (ArC), 160.4
(ArC), 167.7 (C=O), 175.2 (C=O).−
Anal. Calcd for C16H15NO3 (269.30) C 71.29, H 5.57;
Found: C 71.35, H: 5.54.−
4-Methoxy-N-phenylacetylbenzamide (4-14)
N
O O
HOCH
3
Phenylacetic acid amide (4-6a) (1.35 g, 10 mmol) was reacted with 4-methoxybenzoic acid
chloride (1.26 g, 9.4 mmol) as described in procedure A to afford imide 4-14 (807 mg, 30 %),
mp: 162–164 °C.−
IR (KBr)
ν
~ = 3462 cm-1 (N-H), 1786 (C=O), 1601 (C=O).−
Experimental Part
113
1H-NMR (200 MHz, CDCl3): δ = 3.88 (s, 3H, OCH3), 4.37 (s, 2H, PhCH2), 6.95 (t, J = 5 Hz,
2H, ArH), 7.37 (m, 5H, ArH), 7.60 (d, J = 4 Hz, 2H, ArH), 7.86 (d, J = 4 Hz, 2H, ArH), 8.12
(m, 5H, ArH), 9.45 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 39.3 (CH2), 56.0 (OCH3), 114.2 (ArC), 125.8 (ArC), 127.4
(ArC), 128.3 (ArC), 129.0 (ArC), 129.8 (ArC), 135.9 (ArC), 165.4 (ArC), 167.7 (C=O), 170.7
(C=O).−
Anal. Calcd for C16H15NO3 (269.30): C 71.29, H 5.57;
Found: C 71.34, H: 5.54.−
4-(N´-Benzoylhydrazino)-4-oxobut-2-enoic acid ethyl ester (4-16)
NN
O H
H O
OCH
2
CH
3
O
The potassium salt of benzoylhydrazine was prepared by reaction of benzoylhydrazine
hydrochloride (300 mg, 2.2 mmol) in a hot methanolic solution (5 mL) of KOH (98 mg) and
evaporation of the methanol at reduced pressure. The residue was dissolved in CH2Cl2 (10
mL) and fumaric acid chloride ethyl ester (360 mg, 2.7 mmol) was added. The solution was
stirred at room temperature for 1 hour, the precipitate was filtered off and washed with cold
CH2Cl2 to yield 4-16 (256 mg, 50 %), mp: 236–238 °C.−
IR (KBr)
ν
~
= 3498 cm-1 (N-H), 3390 (N-H), 1654 (C=O), 1650 (C=O).−
1H-NMR (200 MHz, DMSO): δ = 1.26 (t, 3H, CH3), 4.20 (q, 2H, CH2), 6.72 (d, J = 15 Hz,
1H, CH), 7.09 (d, J = 15 Hz, 1H, CH), 7.58 (m, 3H, ArH), 7.92 (m, 2H, ArH), 10.53 (s, 1H,
NH), 11.15 (s, 1H, NH).−
13C-NMR (200 MHz, DMSO): δ = 14.9 (CH3), 61.7 (CH2), 128.3 (ArC), 129.4 (ArC), 130.8
(ArC), 131.5 (ArC), 132.7 (ArC), 133.4 (ArC), 135.4 (CH), 137.5 (CH), 161.4 (C=O), 165.6
(C=O), 166.7 (C=O).−
Experimental Part
114
Anal. Calcd for C13H14N2O4: (262.26) C: 59.54, H: 5.38;
Found: C 59.48, H 5.34.−
4-Benzoylaminoxy-4-oxobut-2-enoic acid ethyl ester (4-18)
NO
O
H O
OCH
2
CH
3
O
A mixture of benzoic acid (6.10 g, 50 mmol), hydroxylamine (65 mmol) and dicyclohexyl
carbodiimide (10.30 g, 50 mmol) in methanol (30 mL) was stirred for 1 h. The methanol was
evaporated at reduced pressure and the residue extracted with 10 % aqueous NaOH (10 mL).
The basic phase was acidified with aqueous 10 % HCl (10.3 mL) and extracted with CH2Cl2
(30 mL). The organic phase was dried (Na2SO4) and the solvent removed at reduced pressure.
A sample of this crude hydroxamic acid 4-17 (250 mg), fumaric acid ethyl ester (4-3a) (300
mg, 1.8 mmol), and triethylamine (184 mg, 1.8 mmol) was dissolved in toluene (20 mL) and
stirred for 1 hour at room temperature to yield 4-18 (290 mg, 61 %) after column
chromatography on silica gel, mp: 76–78 °C.−
IR (KBr):
ν
~
= 3076 cm-1 (N-H), 1796 (C=O), 1720 (C=O), 1634 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 1.28 (t, 3H, CH3), 4.24 (q, 2H, CH2), 6.84 (d, J = 13 Hz,
1H, CH), 6.98 (d, J = 13 Hz, 1H, CH), 7.48 (m, 2H, ArH), 7.59 (m, 1H, ArH), 8.09 (m, 2H,
ArH), 9.13 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 14.4 (CH3), 61.9 (CH2), 128.8 (ArC), 129.3 (ArC), 130.5
(ArC), 131.7 (ArC), 132.4 (ArC), 133.5 (ArC), 135.5 (CH), 137.5 (CH), 162.7 (C=O), 169.8
(C=O), 172.3 (C=O).−
Anal. Calcd for C13H13NO5: (263.25): C 59.31, H 4.98;
Found: C 60.03, H 5.03.−
Experimental Part
115
3-(N´-Phenylhydrazinocarbonyl)-acrylic acid ethyl ester (4-20a)
NNOCH2CH3
H O
OH
A mixture of phenylhydrazine (1.08 g, 10 mmol) and fumaric acid chloride ethyl ester (1.34 g,
10 mmol) in dichloromethane (25 mL) and 2−3 drops of triethyl amine was stirred for 30
minutes. The precipitate was filtered off and washed with CH2Cl2 to yield 4-20a (976 mg,
48 %), mp: 140–142 °C.−
IR (KBr):
ν
~ = 3368 cm-1 (N-H), 3096 (N-H), 1644 (C=O), 1614 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 1.27 δ (t, J = 7 Hz, 3H, CH3), 4.22 (q, J = 7 Hz, 2H, CH2),
6.77 (d, J = 14 Hz, 1H, CH), 6.96 (m, 2H, ArH), 7.27 (d, J = 14 Hz, 1H, CH), 7.49 (m, J = 8
Hz, 3H, ArH), 9.98 (s, 1H, NH), 10.15 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 13.8 (CH3), 61.4 (OCH2), 113.2 (ArC), 113.3 (ArC), 119.1
(ArC), 130.0 (ArC), 131.2 (ArC), 135.1 (CH), 137.8 (CH), 144.6 (ArC), 165.2 (C=O), 166.4
(C=O).−
Anal. Calcd for C12H14N2O3: (234.25): C 61.53, H 6.02;
Found: C 61.60, 6.08.−
3-[N´-(2,4-dinitrophenyl)-hydrazinocarbonyl]-acrylic acid ethyl ester (4-20b)
NNOCH
2
CH
3
H O
OH
NO
2
O
2
N
A mixture of 2,4-dinitrophenylhydrazine (1.5 g, 7.6 mmol) and fumaric acid chloride ethyl
ester (1.13 g, 8.4 mmol) in dichloromethane (30 mL) and 2−3 drops of triethyl amine was
stirred for 30 minutes. The precipitate was filtered off, and washed with CH2Cl2 to yield
4-20b (604 mg, 78 %), mp: 161−163 °C.−
Experimental Part
116
IR (KBr)
ν
~ = 3278 cm-1 (N-H), 3024 (N-H), 1695 (C=O), 1670 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 1.28 δ (t, J = 7 Hz, 3H, CH3), 4.24 (q, J = 7 Hz, 2H, CH2),
6.75 (d, J = 14 Hz, 1H, CH), 7.16 (d, J = 14 Hz, 1H, CH), 7.56 (s, 1H, ArH), 8.37 (m, 2H,
ArH), 10.08 (s, 1H, NH), 10.28 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 14.8 (CH3), 61.8 (OCH2), 110.0 (ArC), 113.5 (ArC), 134.4
(ArC), 135.2 (CH), 137.6 (CH), 143.5 (ArC), 148.9 (ArC), 150.1 (ArC), 165.0 (C=O), 166.1
(C=O).−
Anal. Calcd for C12H12N4O7: (324.25): C 44.45, H 3.73;
Found: C 44.36, 3.69.−
3-(N´-phenyl)-hydrazinocarbonyl-acrylic acid octyl ester (4-20c)
NNOC
8
H
15
H O
OH
A solution of phenylhydrazine (504 mg, 4.6 mmol) and fumaric acid monooctyl ester
anhydride (1.15 g, 5.0 mmol) in CH2Cl2 (10 mL) and 2-3 drops of triethyl amine was stirred
at room temperature for 1 hour. The solution was cooled to 0 °C, the precipitate was filtered
off and washed with cold CH2Cl2 to yield hydrazide 4-20c (800 mg, 57 %), mp: 130−132 °C.
IR (KBr):
ν
~
= 3383 cm-1 (N-H), 3283 (N-H), 1675 (C=O), 1594 (C=O).−
1H-NMR (200 MHz, CDCl3): δ = 0.91 (t, 3H, CH3), 1.32 (m, 10H, 5 x CH2), 4.23 (t, J = 4 Hz,
2H, CH2), 6.73 (d, J = 14 Hz, 1H, CH), 7.01 (d, 2H, ArH), 7.21 (d, J = 14 Hz, 1H, CH), 7.51
(m, 3H, ArH), 9.88 (s, 1H, NH), 10.11 (s, 1H, NH).−
13C-NMR (200 MHz, CDCl3): δ = 13.9 (CH3), 22.8 (CH2), 26.1 (CH2), 29.8 (CH2), 30.2
(CH2), 30.5 (CH2), 32.1 (CH2), 65.4 (OCH2), 112.9 (ArC), 118.7 (ArC), 129.1 (ArC), 133.8
(CH), 134.0 (CH), 134.2 (ArC), 165.0 (C=O), 165.7 (C=O).−
Experimental Part
117
Anal. Calcd for C12H12N4O7: (324.25): C 68.65, H 8.49;
Found: C 68.05, 8.43.−
Abbreviations
118
9 Abbreviations
abs. absolute
Bn benzyl
Bzl benzoyl
cat. catalyst, catalytic
cc. concentrated
CD circular dichroism
d doublett
Et ethyl
EtOAc ethyl acetate
EtOH ethanol
h. hour(s)
HOMO highest occupied molecule orbital
IR infrared spectra
LUMO lowest unoccupied molecule orbital
m. multiplet
Me methyl
MeOH methanol
min. minute
mp. melting point
MS mass spectrometry
NMR nuclear magnetic resonance spectroscopy
PAC phenanthrenequinone
PE petrolether
Ph phenyl
q quartet
s singlet
t triplet
temp. temperature
THF tetrahydro furan
TLC thin layer chromatography
Literature
119
10 Literature
[1] H. N. Jayaram, K. Gharehbaghi, J. Rieser, K. Krohn, K. D. Paull, Biochem. Biophys.
Res. Commun. 1992, 186, 1600.
[2] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. D. Paull, D. Vistica, C. Hose,
J. Langely, P. Cronise, A. Walgro-Wolfe, M. Gray-Goodrick, H. Campbell, J. Mayo,
M. Boyd, J. Nat. Cancer Inst. 1991, 83, 757.
[3] M. Abeloff, J. Armitage, J. Niedehuber, Clinical Oncology, 3rd. ed., Churchill
Livingstone, New York 2004.
[4] F. Macdonald, C. H. J. Ford, A. G. Casson, Molecular biology of cancer, 2nd. ed.,
Taylor and Francis, New York 2004.
[5] E. Matutes, N. Parry-Jones, V. Brito-Babapulle, A. Wotherspoon, R. Morilla, R.
Atkinson, M. O. Elnenaei, P. Jain, G. M. Giustolisi, R. P. A'hern, D. Catovsky,
Leukemia and Lymphoma 2004, 45(10), 2007−2015.
[6] A. D. S. Davies, Medical perspectives in cancer research (Ed.: Anthony D. S.
Davies), VCH Verlag, Weinheim 1985.
[7] Renato Baserga, Cell proliferation, cancer, and cancer therapy (Ed.: Renato
Baserga), New York 1982.
[8] J. F. R. Kerr, A. H. Wyllie, A. R. Currie, J. Cancer Research 1972, 26, 239.
[9] J. R. Sulston, H. R. Horvitz, Dev. Biol. 1977, 56, 110.
[10] T. J. McDonnell, G. Numez, F. M. Platt, Mol. Cell Biol. 1990, 10(5), 1901−1907.
[11] M. Michikawa, S. Kikuchi, Kim S. U., Neurosci. Lett. 1992, 8(1), 75−77.
[12] T. Rajkumar, Current Science 2001, 81(5), 535−541.
[13] S. Bunk, The Scientist 2000, 14(12), 23.
[14] K. Gharehbaghi, A. Sreenath, Z. Hao, K. D. Paull, T. Szekeres, D.A. Cooney, K.
Krohn, H. N. Jayaram, Biochem. Pharmacol. 1994, 48, 1413.
[15] H. N. Jayaram, R. L. Dion, R. I. Glazer, D. G. Johns, R. K. Robins, P. C. Srivastava,
D. A. Cooney, Biochem. Pharmacol. 1982, 31, 2371.
Literature
120
[16] D. A. Cooney, H. N. Jayaram, G. Gebeyehu, C. R. Betts, J. A. Kelly, V. E. Marquez,
D. E. Johns, Biochem. Pharmacol. 1982, 31, 2133.
[17] D. A. Cooney, H. N. Jayaram, G. Gebeyehu, J. A. Kelly, V. E. Marquez, D. E. Johns,
G. S. Ahluwalia, R. L. Dion, R. K. Robins, Biochem. Pharmacol. 1982, 32, 2633.
[18] R. C. Jackson, H. P. Morris, G. Weber, Biochem. J. 1977, 1, 166.
[19] C. Simons, Nucleoside Mimetics Their Chemistry and Biological Properties (Ed.:
Gordon and Breach Science Publishers), Australia, Canada 2001.
[20] K. Peasley, Medical Hypotheses 2000, 55, 408.
[21] L. B. Townsend, Chemistry of Nucleosides and Nucleotides, Plenum Press: New
York 1994, 421−535.
[22] A. E. Shaban, A. Z. Nasr, Adv. Heterocycl. Chem. 1998, 71, 164.
[23] K. Krohn, H. Heins, K. Wielckens, J. Med. Chem. 1992, 35, 511−517.
[24] R. Barker, H. G. Flechter, J. Org. Chem. 1961, 26, 4605.
[25] R. R. Schmidt, J. Karg, W. Guilliard, Chem. Ber. 1977, 110, 2433−2444.
[26] W. Timpe, K. Dax, N. Wolf, H. Weidmann, Carbohydrate Research 1975, 39, 58.
[27] A. I. Meyers, D. L. Temple, D. Haidukewych, E. D. Mihelich, J. Org. Chem 1974,
39, 2787.
[28] W. Liu, J. A. Walker, Jiong J., Wise, D. S. Chen, L. B. Townsend, Tetrahederon
Letters 1996, 37(30), 5325−5328.
[29] W. Liu, D. S. Wise, L. B. Townsend, J. Org. Chem. 2001, 66(14), 4783−4786.
[30] W. Theodora Greene, Peter G. M. Wuts, Protective Groups in Organic Synthesis in
Protection for the Carboxyl Group (Eds.: W. Theodora Greene, Peter G. M. Wuts),
3rd. ed., chapter 5, JOHN WILEY & SONS, INC., New York, pp. 369−454.
[31] C. Schotten, E. Baumann, Ber. 1884, 17, 2544; Ber. 1886, 19, 3218.
[32] R. M. DeLederkremer, M. I. Litter, L. F. Sala, Carbohydrate Res. 1974, 36, 185.
[33] M. Okabe, R. Chu-Sun, G. B. Zenchoff, J. Org. Chem. 1991, 56(14), 4392−4397.
[34] E. J. Enholm, S. Jiang, K. Abboud, J. Org. Chem. 1993, 58(15), 4061−4069.
[35] Z-X. Wang, I. L. Wiebe, E. De Clercq, J. Balzarini, E. E. Knaus, Can. J. Chem.
2000, 78, 1081−1088.
Literature
121
[36] A. Choudhury, F. Jin, D. Wang, Z. Wang, G. Xu, D. Nguyen, J. Castoto, M. E.
Pierce, P. N. Cofalone, Tetrahedron Letters 2003, 44, 247−250.
[37] U. Ravid, R. M. Silverstein, L. R Smith, Tetrahedron 1978, 34(10), 1449−1452.
[38] M. Taniguchi, K. Koga, S. Yamada, Tetrahedron 1974, 30, 3547−3552.
[39] R. R. Schmidt, Jung K.-H., Trichloroacetimidates in Carbohydrates in Chemistry
and Biology in Part I: Chemistry of Saccharides (Eds.: B. Ernst, G. W. Hart, P.
Sinaÿ), 1st. ed., vol. 1, chapter 1, Wiley-VCH, Weinheim 2000, pp. 5−59.
[40] A. Chiou, G. Kokotos, Synthesis 1998, 2, 168−170.
[41] Daniele Hoffmann, David B. Collum, J. Am. Chem. Soc. 1998, 120, 5810−5811.
[42] V. Derdau, V. Snieckus, J. Org. Chem. 2001, 66(6), 1992−1998.
[43] K. Gharehbaghi, A. Sreenath, Z. Hao, K. D. Paull, T. Szekeres, D. A. Cooney, A.
Monks, D. Scudiero, Krohn. K., H.N. Jayaram, Int. J. Cancer 1994, 56, 892.
[44] J. Ludwig, Acta Biochim. et Biophys. Acad. Sci. Hung. 1981, 16(3−4), 131−133.
[45] G. Gebeyehu, V. E. Marquez, J. A. Kelley, D. A. Cooney, H. N. Jayaram, D. G.
Johns, J. Med. Chem. 1983, 26, 922−925.
[46] G. Gebeyehu, V. E. Marquez, A. van Cott, D. A. Cooney, J. A. Kelly, H. N. Jayaram,
G. S. Ahluwalia, L. R. Dion, Y. A. Wilson, A. G. Johns, J. Med. Chem. 1985, 28,
99−105.
[47] A. Zatorski, K. A. Watanabe, S. F. Carr, B. M. Goldstein, K. W. Pankiewicz, J. Med.
Chem. 1996, 39, 2422−2426.
[48] K. Krohn, C. Franke, P. G. Jones, H.-J. Aust, S. Draeger, B. Schulz, Liebigs Ann.
Chem. 1992, 789−798.
[49] K. Krohn, A. Michel, U. Flörke, H.−J. Aust, S. Draeger, B. Schulz, Liebigs. Ann.
Chem. 1994, 1093−1097.
[50] Y. Suhara, H. B. Maruyama, Y. Kotoh, Y. Yokose, K. Miyasaka, H. Shirai, K.
Takano, J. Antibiotics 1975, 28, 648−655.
[51] J. Cason, J. B. Miller, J. Am. Chem. Soc. 1942, 1106−1110.
[52] B. Talapatra, A. Mukhopadhayay, Indian J. Chem. 1982, 21 B, 386−387.
[53] H. Y. Chen, M. S. Shiao, Y. L. Huang, J. Nat. Prod. 1999, 62, 1225−1227.
Literature
122
[54] T. H. Lin, S.J. Chang, C.C. Chen, J. Nat. Prod. 2001, 64, 1084−1086.
[55] H. W. Schmalle, B. M. Hausen, Naturwiss 1979, 66, 527−528.
[56] T. S. Wu, T. T. Jong, H. J. Tien, Phytochemistry 1987, 26, 1623−1625.
[57] A. C. Alder, P. Rüedi, C. H. Eugster, Helv. Chim. Acta 1984, 67, 1003−1011.
[58] A. C. Alder, P. Rüedi, R. Prew, Helv. Chim. Acta 1986, 69, 1395−1417.
[59] S. B. Singh, G. R. Pettit, J. Org. Chem. 1989, 54, 4105−4114.
[60] T. O'Criodain, B. O'Sullivan, M. J. Meegan, Phytochem. 1981, 20, 1089−1092.
[61] D. M. X. Donnelly, Proc. Int. Bioflav. Symp. Munich 1981, 263−268.
[62] O. Edwards, G. Feniak, M. Los, Canad. J. Chem. 1962, 40, 1540−1546.
[63] J. H. Gough, M. D. Sutherland, Austr. J. Chem. 1966, 19, 329−330.
[64] J. Reisch, M. Bathory, I. Novak, Herba Hung. 1972, 11, 61−71.
[65] C. H. Brieskorn, L. Buchberger, Planta med. 1973, 24, 190−195.
[66] M. Onitsuka, M. Fujiu, N. Shinma, Chem. Pharm. Bull. 1983, 31, 1670−1675.
[67] H. M. Chang, K. P. Cheng, T. F. Choang, J. Org. Chem. 1990, 55, 3537−3543.
[68] S. M. Kupchan, A. Karim, C. Marks, J. Org. Chem. 1969, 34, 3912−3918.
[69] H. H. Babcock, The Pharmacist (Chicago) 1875, 1, 8.
[70] D. T. MacDougal, Minnesota Bot. Stud. 1894, 1, 32−36.
[71] D. T. MacDougal, Minnesota Bot. Stud. 1895, 2, 450−451.
[72] A. Nestler, Bericht. dtsch. bot. Ges. 1907, 23, 554−556.
[73] H. Beierlein, Orchidee 1957, 8, 95.
[74] B. M. Hausen, Orchidee 1978, 29, 134.
[75] B. M. Hausen, Arch. Dermatol. 1980, 116, 327−328.
[76] B. M. Hausen, Arch. Derm. Res. 1979, 254, 102−103.
[77] B. M. Hausen, Toxic and allergenic orchids in Orchid. Biology (Ed.: Cornell
University Press), 3rd. ed., London 1984, 263−282.
[78] B. M. Hausen, Woods injurious to human health (Ed.: de Gruyter), Berlin, New York
1981.
[79] R. J. Schmidt, L. Fernandez de Corres, Contact Dermatitis 1990, 23, 260−261.
[80] D. M. Owili, East Afric. Med. J. 1977, 54, 571−573.
Literature
123
[81] G. Hopff, H. R. Schweizer, Helv. Chim. Acta 1963, 45, 312−331.
[82] M. S. Newman, R. L. Childers, J. Org. Chem. 1967, 32, 62−66.
[83] B. M. Hausen, H. Heitsch, B. Borrmann, Contact Dermatitis 1995, 33, 12−16.
[84] K. Krohn, U. Loock, K. Paavilainen, B. M. Hausen, H. W. Schmalle, H. Kiesele,
ARKIVOC 2001, 2(7), 973−1003.
[85] C Schwarzkopf, B. Thiele, http://www.zet.or.at/publikationen/buecher/band-
iv/schwarzkopf.htm.
[86] I. Fleming, Frontier Orbitals and Organic Reactions (Ed.: John Wiley & Sons),
London 1976.
[87] P. Schreier, M. Herderich, H-U. Humpf, W. Schwab, Natural Product Analysis in
The quantumchemical calculation of CD spectra: the absolute configuration of
chiral compounds from natural or synthetic origin (Ed.: Lengerich Lengericher
Handelsdruckerei), 1.st. ed., Vieweg and Sohn, Braunschweig/Wiesbaden 1998, pp.
195−220.
[88] N. Harada, K. Nakanishi, J. Am. Chem. Soc. 1975, 97, 5345−5352.
[89] K. Nakanishi, N. Berova, R. W. Woody, Circular Dichroism (Ed.: Weinheim VCH
Verlagsgesellschaft mbH), VCH Verlagsgesellschaft mbH, Weinheim 1994.
[90] N. Harada, K. Nakanishi, Circular Dichroic Spectroscopy - Exciton coupling in
organic stereochemistry (Ed.: University Science books), Oxford University Press,
Oxford 1983.
[91] L. Rosenfeld, Z. Phys. 1929, 52, 161−174.
[92] J. W. Downing (Department of Chemistry and Biochemistry, University of Colorado,
Boulder), Programm Package BDZDO/MCDSPD.
[93] P. S. Steyn, Tetrahedron 1970, 26, 51−57.
[94] M. Isaka, A. Jatuparat, K. Rukseree, K. Danwisetkanja, M. Tanticharoen, Y.
Thebtatanonth, J. Nat. Prod. 2001, 64, 1015−1018.
[95] I. Kurobane, L. C. Vining, A. G. McInnes, Tetrahedron Letters 1978, 47,
4633−4636.
Literature
124
[96] Michinori Oki, Topics in Stereochemistry in Recent Advances in Atropisomerism,
vol. 14, Wiley, New York 1983, pp. 1−76.
[97] E. L. Eliel, S. H. Wilen, Stereochemistry of organic compounds, chapter 14−15, John
Wiley & Sons Inc., New York 1994, pp. 1142−1155.
[98] G. Bringmann, J. Hinrichs, P. Henschel, J. Kraus, K. Peters, E−M. Peters, Eur. J.
Org. Chem. 2002(6), 1096−1106.
[99] O. Hofer, R. Schölm, Tetrahedron 1981, 37, 1181−1186.
[100] R. S. Ward, Natural product reports 1997, 14(1), 43−74.
[101] A. A. Ahmed, A. A. Mahmoud, E. T. Ali, O. Tzakou, M. Couladis, T. J. Mabry, T.
Gáti, Tóth G., Phytochemistry 2002, 59, 851−856.
[102] I. R. Bytheway, E. L. Ghisalberti, S. Gotsis, P. R. Jefferies, B. W. Skelton, K. E.
Sugars, A. H. White, Aust. J. Chem. 1987, 40, 1913−1917.
[103] Y. Lin, S. Wu, S. Feng, G. Jiang, J. Luo, S. Zhou, L. L. P. Vrijmoed, E. B. G. Jones,
K. Krohn, K. Steingroever, F. Zsila, Tetrahedron 2000, 57, 4343−4348.
[104] S. Antus, G. Snatzke, I. Steinke, Liebigs. Ann. Chem. 1983, 2247.
[105] K. Krohn, R. Bahramsari, U. Flörke, K. Ludewig, C. Kliche-Spory, A. Michel, H-J.
Aust, S. Draeger, B. Schulz, S Antus, Phytochemistry 1997, 45(2), 313−320.
[106] T. K. Shing, J. Yang, J. Org. Chem. 1995, 60(18), 5785−5789.
[107] J. P. Jennings, W. Klyne, P. M. Scopes, J. Chem. Soc. 1965, 7211−7229.
[108] G. Albers Scönberg, H. Schmidt, Helv. Chim. Acta 1961, 44(181), 1447−1471.
[109] F. Abe, R. F. Chen, T. Yamauchi, Chem. Pharm. Bull. 1988, 36(8), 2784−2789.
[110] H. Franzyk, Synthetic Aspects of iridoid Chemistry in Prog. Chem. Nat. Prod. (Eds.:
W. Herz, FalkH., G. W. Kirby), vol. 83, Sprenger, Wien - New York 2000, pp.
2−104.
[111] G. E. Martin, R. Sanduja, M. Alam, J. Org. Chem 1985, 15(50), 2383−2386.
[112] B. M Trost, J. M. Balkovec, M, K-T. Mao, P. Buhlmayer, J. Am. Chem. Soc. 1986,
108(16), 4965−4973; J. Am. Chem. Soc. 1986, 108(16), 4973−4983.
Literature
125
[113] A. D. Lighter, E. J. Gurst, Organic Conformational Analysis and Stereochemistry
from Circular Dichroism Spectroscopy (Ed.: John Wiley and Sons Inc.), Wiley-
VCH, New York 2000.
[114] G. A. Kraus, J. Thurston, J. Am. Chem. Soc. 1989, 111(26), 9203−9205.
[115] Autorenkollektiv, Organikum (Ed.: VEB Deutscher Verlag der Wissenschaften),
18st. ed., Berlin 1990, pp. 290−291.
[116] D. D. Perin, W. L. F. Armarego, D. R. Perin, Purification of Laboratory Chemicals
(Ed.: Pergamon Press), 2nd. ed., Oxford, New York.
[117] Z.-X. Wang, L. I. Wiebe, E. De Clercq, J. Balzarini, E. E. Knaus, Can. J. Chem
2000, 78(8), 1081−1088.
[118] H. S. Jensen, G. Limberg, C. Pedersen, Carbohydrate Res. 1997, 302, 109−112.
[119] D. R. Dalton, Y. Huang, J. Org. Chem. 1997, 372−376.
[120] J. Hajdu, N. S. Chandrakumar, J. Org. Chem. 1982, 2144−2147.
