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Exploration of pathways for the synthesis of dimethyl-1,4-diamino-1,4-cyclohexane dicarboxylate, a novel bis-amino acid ester

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Exploration of pathways for the synthesis of dimethyl-1,4-diamino-1,4-cyclohexane dicarboxylate, a novel bis-amino acid ester
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Dashzeveg, Rentsenmyadag
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English
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74 leaves : illustrations ; 28 cm

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Esters -- Synthesis ( lcsh )
Esters -- Synthesis ( fast )
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theses ( marcgt )
non-fiction ( marcgt )

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Includes bibliographical references (leaf 74).
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by Rentsenmyadag Dashzeveg.

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University of Colorado Denver
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Full Text
EXPLORATION OF PATHWAYS FOR THE SYNTHESIS OF DIMETHYL-1,4-
DIAMINO-1,4-CYCLOHEXANE DICARBOXYLATE, A NOVEL BIS-AMINO
ACID ESTER
by
Rentsenmyadag Dashzeveg
B.S., Mongolian National University, 1994
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
Chemistry
2003


This thesis for the Master of Science
Degree by
Rentsenmyadag Dashzeveg
has been approved
by
Douglas F. Dyckes
Donald G,Zapien
Ju.rU,
Date
Doris R. Kimbrough


i
i
Rentsenmyadag Dashzeveg (M.S., Chemistry)
Exploration of Pathways for the Synthesis of Dimethyl-1,4-diamino-1,4-cyclohexane
Dicarboxylate, a Novel Bis-amino Acid Ester
Thesis directed by Professor Douglas F. Dyckes
ABSTRACT
The essential goal of this research was to investigate a pathway to synthesize
dimethyl-1,4-diamino-1,4-cyclohexane dicarboxylate, a novel bis-amino ester. The
synthesis of 1,4-diformylamino-1,4-cyclohexane dicarboxylic acid was optimized,
with a yield of 87-90%. Formylation was carried out to increase solubility of the bis-
amino acid while providing protection for the amino functions. The formylated bis-
amino acid was used as a starting material for many esterification attempts.
The first attempted pathway for esterification, standard Fischer esterification,
was without success. The major difficulty during previous esterification attempts had
been the insolubility of the underivatized bis-amino acid under esterification
conditions. When the formylated compound was used, the acidic conditions required
for esterification apparently cleaved the formyl group before the formation of ester
could occur, and the reaction failed.
A second attempted pathway for esterification was direct methylation of the
carboxylate group by CH3I. The methylation reactions were carried out under several
conditions, using different solvents (DMF or DMSO) and different bases (K2CO3 or
Et3N). All failed.
111


A third proposed pathway for esterification was explored using DCC as a
coupling reagent. Normally DCC is highly reactive and gives high yields of esters
within a short time. The three different conditions for esterification tested using DCC
as a coupling reagent all failed.
The fourth pathway attempted for esterification used SOCI2 to activate the
carboxyl group. This method was tried on both the formylated and the unprotected
bis-amino acid, using a DMSO/SOCb/MeOH system. Neither reaction gave clear
positive results.
A very different pathway for esterification was investigated, using a precursor
of the bis-amino acid. 1,4-Diamino-1,4-dicyanocyclohexane was reacted with sodium
methoxide in an attempt to convert the nitrile directly into the ester.
The sixth proposed pathway to the bicyclic amide avoids esterification. It is a
thermal condensation reaction of 1,4-diamino-1,4-cyclohexanedicarboxylic acid.
Heating liberated water and permanently modified the bis-amino acid, but the bicyclic
monomer could not be isolated from the product.
This abstract accurately represents the content of the candidates thesis.
I recommend its publication.
Signed
IV
Douglas F. Dyckes


ACKNOWLEDGEMENTS
My special thanks to Professor Douglas Dyckes for his support, guidance and
patience throughout this work.
I also wish to express my gratitude to Professor Donald Zapien and Professor Doris
Kimbrough for their advice and assistance.
v


CONTENTS
I
I
Figures.................................................................. xi
Schemes................................................................ xiii
Tables.................................................................. xiv
Structures of Compounds Described in this Work........................... xv
Chapter
1. Introduction........................................................... 1
1.1 Research Goal......................................................... 1
1.2 Background............................................................ 5
1.3 Synthetic Strategy.................................................... 5
1.3.1 Protection of the Amino Group of an Amino Acid.................. 6
1.3.2 Activation of the Carboxyl Group of an Amino Acid............... 6
2. Results and Discussion............................................. 11
2.0. 1 Synthesis of 1,4-Diamino-1,4-dicyano cyclohexane (Compound I).. 11
2.0. 2 Synthesis of 1,4-Diamino-1,4-cyclohexane Dicarboxylic Acid 13
(Compound II)....................................................
2.1 Optimization of the Amino Protection of Compound II by Formylation... 17
2.2. First Attempted Synthesis of Compounds IV and V:
vi
Standard Fischer Esterification
22


2.2.1 Attempted Fischer Esterification of Compound II..................... 23
2.2.2 Attempted Fischer Esterification of Compound HI..................... 26
2.3 Attempted Esterification with Methyl Iodide.......................... 29
2.3.1 Attempted Methylation of Compound III in DMF using K2CO3 as
the Base ............................................................... 31
2.3.2 Attempted Methylation of Compound III in DMSO using K2CO3 as the
Base.................................................................... 32
2.3.3 Attempted Methylation of Compound III in DMSO using an Organic
Base, Triethylamine..................................................... 32
2.4 Third Proposed Pathway to Compound HI: Esterification Using DCC as a
Coupling Reagent.......................................................... 34
2.4.1 Attempted Esterification of Compound III using DCC and Methanol in
DMSO.................................................................... 36
2.4.2 Attempted Esterification of Compound III using DCC and Para-
nitrophenol in DMSO...................................................... ^
2.4.3 Using DCC as a Coupling Reagent (Para-nitrophenol at 70 C)........ 39
2.5 Attempted Esterification Using SOCI2..................................... 40
2.5.1 Attempted Esterification of Compound II in Methanol using Thionyl
Chloride................................................................ 41
vii


2.5.2 Attempted Esterification of Compound III using Thionyl Chloride in
DMSO............................................................ 43
2.6. Attempted Methanolysis of the Nitrile Functions of Compound 1. 44
2.6.1 Attempted Methanolysis of Compound I in Acidic Conditions...... 44
2.6.2 Attempted Methanolysis of Compound I in Basic Conditions....... 47
2.7 Attempts to Cyclize Compound II by Heating....................... 49
2.7.1 Attempt to Cyclize Compound 13 by Heating with a Torch......... 49
2.7.2 Attempt to Cyclize Compound II by Heating in a Heating Block. 50
2.8 Conclusions...................................................... 51
3. Experimental...................................................... 53
3.1 Reagents......................................................... 53
3.2 General Methods.................................................. 53
3.2.1 Thin Layer Chromatography...................................... 53
3.2.2 Nuclear Magnetic Resonance Spectroscopy........................ 54
3.2.3 Infrared Spectroscopy.......................................... 54
3.3 Experimental Procedures........................................ 55
3.3.1 Synthesis of l,4-Dicyano-l,4-diaminocyclohexane (Compound I). 55
3.3.2 Synthesis of 1,4-Diamino-1,4-cyclohexane Dicarboxylic Acid
(Compound II).................................................... 56
viii


3.3.3 Synthesis of 1,4-Diformylamino of 1,4-cyclohexane Dicarboxylic Acid
(Compound HI)...................................................... 57
3.3.4 Attempted Synthesis of 1,4-dimethyl-1,4-diamino Cyclohexane
Dicarboxylate (Compound IV) Using Fischer Esterification........... 58
3.3.5 Attempted Synthesis of Dimethyl-1,4- diformylamino-1,4-cyclohexane
Dicarboxylate (Compound V) using Fischer Esterification............ 59
3.3.6 Attempted Synthesis of Compound V using Methyl Iodide and K2CO3
in DMF............................................................. 60
3.3.7 Attempted Synthesis of Compound V using Methyl Iodide and K2CO3
in DMSO............................................................ 61
3.3.8 Attempted Synthesis of Compound V using Methyl Iodide and
Triethylaminein DMSO............................................... 62
3.3.9 Attempted Synthesis of Compound V in DMSO using DCC as
an Activating Agent................................................ 64
3.3.10 Attempted Synthesis of Bis-nitrophenyl-l,4-diformylamino-1,4-
cyclohexane dicarboxylate using DCC as an Activating Agent...... 65
3.3.11 Second Attempted Synthesis of Bis-para-nitrophenyl-1,4-
diformylamino- 1,4-cyclohexane dicarboxylate using DCC as an
Activating Agent at Elevated Temperature........................... 66
IX


3.3.12 Attempted Synthesis of Compound IV using SOCI2 in
Methanol......................................................... 67
3.3.13 Attempted Synthesis of Compound V using SOCI2 in DMSO....... 68
3.3.14 Attempted Synthesis of Compound IV by Methanolysis of
Compound I in Acidic Conditions................................. 69
3.3.15 Attempted Synthesis of Compound IV by Methanolysis of
Compound I in Basic Conditions.................................. 70
3.3.16 A Heating Reaction of Compound II Using a Torch............. 72
3.3.17 A Heating Reaction of Compound II Using a Heating Block....... 73
References............................................................. 74
x


FIGURES
Figure 1.1 Dimethyl-1,4-diamino-1,4-cyclohexane dicarboxylate.......... 1
Figure 1.2 Schematic drawing of the formation and polymerization of a
bicyclic monomer from dimethyl-1,4-diamino
-1,4-cyclohexane dicarboxylate............................... 2
Figure 1.3 Schematic drawing of the polymerization of the dimethyl-
1,4-diamino-1,4-cyclohexane dicarboxylate as a spiropolymer
of cyclohexane and DKP units.................................. 3
Figure 2.0.1 IR spectrum of Compound 1.................................. 12
Figure 2.0.2 X-ray diffraction structure of Compound II................. 14
Figure 2.0.3 IR spectrum of Compound II................................. 16
Figure 2.1.1 'H-NMR spectrum at 200 MHz of Compound III in
D20/ NaOD................................................... 19
Figure 2.1.2 IR spectrum of Compound HI................................. 21
Figure 2.2.1 1 H-NMR spectrum at 200 MHz of the residue from
attempted Fischer esterification of Compound 13............. .
Figure 2.2.2 1 H-NMR spectrum in d-methanol at 200 MHz of the residue
from attempted Fischer esterification of Compound III......................
Figure 2.4.1 'H-NMR spectrum in d6-acetone at 200MHZ of the product
from treating Compound HI with DCC.......................................
38


Figure 2.5.1 'H-NMR spectrum D20/ NaOD at 200 MHz of the product from
treating Compound II with SOCl2 in methanol.................. 42
Figure 2.6.1 'H-NMR spectrum in d- methanol at 200 MHz of the product
from treating Compound I with methanol in acidic conditions... 46
Figure 2.6.2 'H-NMR spectrum in d^-acetone at 200 MHz of the product from
treating Compound I with methanol in basic conditions........ 48


SCHEMES
Scheme 1: Proposed routes for the esterification of Compound III.............. 8
Scheme 2: Proposed routes to the synthesis of Compound IV by
methanolysis of Compound 1............................................... 9
Scheme 3: Proposed direct cyclization of Compound II by heating................. 10
Scheme 4: Synthesis of N-formyl derivatives..................................... 18
Scheme 5: The attempts at esterification of Compound 13 and Compound
HI using Fischer esterification......................................... 22
Scheme 6: The attempts at esterification of Compound IH using methyl
iodide.................................................................. 30
Scheme 7: Proposed esterifications of the carboxyl group of Compound in
by DCC.................................................................. 35
Scheme 8: The attempts at esterification of Compound II and Compound HI
using SOCI2............................................................. 40
xm


TABLES
2.0. 1 Elemental Analysis of Compound 1....................... 11
2.0. 2 Elemental Analysis of Compound II...................... 13
1
I
!
i
i
XIV


STRUCTURES OF COMPOUNDS DESCRIBED IN THIS WORK
Compound I:
Compound II:
Compound ID :
Compound IV :
Compound V :
h2n,cn
$
NC NH2
H2N COOH
0
HOOC NH2
H-CO-NH .COOH
HOOC NH-CO-
H
CH3-O-OC NH2
H-CO-NH CO-O-CH3
CH3-O-OC NH-CO-H
XV


1. Introduction
1.1 Research Goal
The essential goal of this research was to investigate a pathway to
synthesize dimethyl-1,4-diamino- 1,4-cyclohexane dicarboxylate a novel
bis-amino acid ester, which could be suitable for peptide and polymer
synthesis. (Fig 1.1)
Figure 1.1 Dimethyl-1,4-diamino-1,4-cyclohexane dicarboxylate
Dimethyl-1,4-diaminocyclohexane-1,4- dicarboxylate could be used in several
ways as a new monomer. It has two separate amino acid units capable of forming
peptide bonds. It could be used to bridge or cyclize synthetic peptide chains.
Dimethyl-1,4 diaminocyclohexane-1,4- dicarboxylate could also be internally
cyclized to form a bicyclic amide. These units might then be reacted to form a
polymer, as shown in Fig 1.2.
1


o
h2n
II
c o ch3
ch3 oc
II
o
NH
2
1 .First step (internal cyclization)
O
O
II
NH-C
CH3 O
NH
2
2. Second step ( Polymerization)
Figure 1.2 Schematic drawing of the formation and polymerization
of a bicyclic monomer from dimethyl-1,4- diamino-1,4-cyclohexane
dicarboxylate
2


Alternatively, if dimethyl-1,4-diamino-1,4-cyclohexane dicarboxylate
polymerized in the diketopiperazine (DKP) form (six membered heterocyclic
rings containing two peptide bonds) the polymer shown in Fig 1.3 would be
obtained.
Figure 1.3 Schematic drawing of the polymerization of dimethyl-1,4-
diamino-1,4-cyclohexane dicarboxylate as a spiropolymer of cyclohexane
and DKP units [----indicates hydrogen bonding ].
3


However, this extended spiro-cyclostructure may be too sterically hindered to be
easily formed. If it can be made, the spiro-cyclo polymer could be interesting because
of the rigidness conferred by both the cyclohexane and DKP rings. The formation of
hydrogen bonds between amide groups of the DKP rings of different polymer strands
could provide additional rigidity. In this research, we concentrated our investigations
on the synthesis of the ester. Many different attempted approaches to the synthesis of
dimethyl-1,4-diamino-1,4-cyclohexane dicarboxylate are reported in this thesis.
4


1.2 Background
Carolina Wilson (1), a former graduate student, successfully synthesized 1,4-
diamino-l,4-dicyanocyclohexane (Compound I) and 1,4-diamino-1,4-cyclohexane
dicarboxylic acid (Compound II). She also tried several different approaches for the
derivatization of Compound II. She was unsuccessful in attempts to add the Fmoc or
trifluoroacetyl protecting groups. These reactions failed because of the insolubility of
Compound II in most reaction media. She was successful in synthesizing the
formylated and acylated forms of Compound II. Acetyl groups can not be removed by
mild conditions. Therefore acetyl group protection is not a useful strategy. Formyl
groups are readily removed by treatment with 0.5 N HC1 at room temperature. Wilson
also tried to synthesize the bis-amino acid ester by using Fischer esterification. All of
these experiments used a single batch of Compound II, and none were successful.
1.3 Synthetic Strategy
Peptides and proteins consist of chains formed by amino acids linked to one
another by amide bonds. Therefore the principal reaction in the building of such
chains is the acylation of the amino group of one amino acid by the carboxyl group of
a second amino acid (2). Since amide bond formation does not occur spontaneously
except at elevated temperatures, one of these two groups (usually the carboxyl group)
has to be converted to a more reactive form (3).
5


1.3.1 Protection of the Amino Group of an Amino Acid
Protection of the amino group of one of the amino acids being combined is
one step of peptide synthesis. These protecting groups must be easily removable after
the peptide bond formation. The formylated compound, 1,4-diformylamino-1,4-
cyclohexane dicarboxylic acid (Compound HI) will be used in this work as an
intermediate in the esterification studies. Formyl protecting groups are removable by
mild acidic conditions, but are stable to basic reaction conditions.
1.3.2 Activation of the Carboxyl Group of an Amino Acid
Formation of a peptide bond requires activation of the carboxyl group of an
amino acid. Activation makes the carbon atom of the carboxyl group sufficiently
electrophilic to facilitate nucleophilic attack by the amino group. Esters are capable of
activating carboxyl groups for some amide-bond-forming reactions.
This thesis concentrates on efforts to make the methyl ester, dimethyl-1,4-diamino-
1,4-cyclohexane dicarboxylate (Compound IV), by a number of different
approaches. Several proposed derivatizations of the carboxyl group are shown in
Scheme 1 (following page).
The following reactions were explored as to esterify Compounds II and III:
1. Standard Fischer esterification ,
2. methylation by methyl iodide,
6


3. esterification by activation using dicyclohexylcarbodiimide (DCC), and
4. thionyl chloride activation of the carboxyl group in the presence of methanol.
7




In addition, a different starting material, Compound I, could be used in efforts to
make Compound IV. These approaches are shown in Scheme 2.
Compound I
Scheme 2: Proposed routes to the synthesis of Compound IV by
methanolysis of Compound I
9


Scheme 3 shows attempts at direct internal cyclization of Compound n. In this
latter approach the synthesis of dimethyl-1,4-diamino-1,4-cyclohexane
dicarboxylate is an attempt to prepare the bicyclic amide directly. If succcessful,
esterification or other activation of the remaining carboxylate group would follow.
COOH
NH3
Scheme 3: Proposed direct cyclization of Compound II by
heating
10


2. Results and Discussion
2.0.1 Synthesis of l,4-Diamino-l,4-dicyano cyclohexane
(Compound I)
Compound I was synthesized as the first step of the Strecker synthesis of
Compound II (1). 1,4-cyclohexanedione was reacted with sodium cyanide and
ammonium chloride in the presence of methanol to form 1,4-diamino-1,4-
dicyanocyclohexane. The elemental analysis of Compound I is shown in Table 2.0.1.
Table 2.0.1: Elemental Analysis of Compound I
Elemental Theory Found
C 58.51 58.63
H 7.37 7.37
N 34.12 33.90
The experimental elemental analysis result was very close to the theoretical values.
The H-NMR spectrum (D2O/DCI) for Compound I is composed two doublets at 2.33
and 2.28 ppm, and at 1.91 and 1.86 ppm. This corresponds to the spectrum obtained
by Wilson (1).
The IR spectrum for Compound I (Fig 2.0.1) shows a clear CN stretching band at
2222 cm'1. Other important absorptions are seen at 3280 cm'1 and 3179 cm'1 (NH2),
and at 2953 cm'1 (Aliphatic CH stretch)
11




2.0.2 Synthesis of 1,4-Diamino-l,4-cyclohexane
Dicarboxylic Acid (Compound I)
The synthesis of Compound II was carried out by hydrolysis of Compound I.
The elemental analysis of Compound II is shown in Table 2.0.2
Table 2.0.2: Elemental Analysis of Compound II
Elemental Theory Theory Found
Compound II Compound II+2H2O
C 47.52 40.33 40.32
H 6.98 7.62 7.45
N 13.85 11.76 11.70
The formula of Compound II is C8H14O4N2. But the elemental analysis result
corresponds closely to CgHisOs^. It matches 1,4-diamino-1,4-cyclohexane
dicarboxylic acid crystallized with 2 molecules of H2O.
X-ray diffraction analysis of Compound II, crystallized from a copper sulfate
solution by Samuel Allen in this laboratory, confirms its structure and shows that the
carboxylate groups (as well as the amino groups) are trans to one another.
Interestingly, the Compound H^SCVCu^O^ crystal unit cell also contains two
non-ligand H2O molecules. These two water molecules are indicated by circles in the
X-ray diffraction structure shown in Fig 2.0.2.
13


Fig 2.0.2 X-ray diffraction structure of Compound II


The 'H-NMR spectrum for Compound II in base (D20/NaOD) is composed of two
doublets at 1.90 and 1.85 ppm, and at 1.35 and 1.31 ppm. The !H-NMR spectrum for
Compound II in acid (D2O/DCI) is composed of a multiplet signal between 1.86-2.03
ppm. These correspond to the spectra obtained by Wilson (1).
The assignment of the IR spectrum (Fig 2.0.3) for Compound II is as follows:
A broad (-NH3+) stretching band in the 3100-2100 cm'1 region, aliphatic C-H stretch,
2920 cm'1, asymmetric carboxylate (C-O) stretch, 1576 cm'1, symmetric carboxylate
(C-O) stretch, 1400 cm'1, symmetric (-NH3+) N-H bend, 1527 cm'1, asymmetric
(NH3+) N-Hbend, 1610 cm'1
15


Fig 2.0.3 IR spectrum of Compound II


2.1 Optimization of the Amino Protection of
Compound II by Formylation
Compound III was synthesized following procedures described by du
Vigneaud et al (4). The first step of the formylation reaction is the formation of the
mixed anhydride between the formic acid and acetic anhydride. Later, nucleophilic
attack of the amino group of Compound II on the formyl group of the mixed
anhydride yields the desired formyl derivative. The reaction for the synthesis of
Compound HI is shown in Scheme 4.
The procedure was optimized by formylating in different solvents: 96%
formic acid (7 times), 99% formic acid diluted to 96% formic acid (2 times) and 99%
formic acid (2 times). The reaction in 96% HCOOH gave a good yield (87-90%), and
good NMR and IR spectra. But the reaction in 99% HCOOH gave low yields. From
these results, it appears that the most convenient and useful medium is 96% HCOOH.
The assignment of the 'H-NMR (DaO/NaOD) spectrum (Fig 2.1.1) for
Compound in is as follows. The singlets at 8.08 and 7.97 ppm, which integrate
together as 2 protons, correspond to the formyl protons in the cis and trans
conformations, respectively. The multiplet between 1.79-2.00 ppm, which integrates
17




7.171
Fig 2.1.1 'll -NMR spectrum at 200 MHz of Compound HI in DjO /NaOD


as 8 protons, corresponds to the protons of the cyclohexane ring. It apparently
consists of two overlapping triplets. (The singlet at 4.8 ppm corresponds to HOD
peak).
The assignment of the IR spectrum (Fig 2.1.2) for Compound III is as follows:
3186 cm'1 (NH stretch), 2888 cm'1 (formyl CH stretch), 1643 cm'1 (formyl CO stretch
and carboxylic acid CO stretch).
20


Fig 2.1.2 JR spectrum of Compound III


2.2 First Attempted Synthesis of Compounds IV
and V: Standard Fischer Esterification
The syntheses of Compound IV and Compound V were atttempted by
Fischer esterification without success. The major difficulty during
esterification reactions was the insolubility of both the compound II and
compound in in acidic conditions. The reaction conditions attempted are
shown in Scheme 5.
O
HOOC NH2
HOOC NH-c------H
1 .CH3OH (excess)
1 .CH3OH (excess)
2.HC1 (g)
2.HC1 (g)
O
CH3-O-OC NH2 CH3-O-OC NH-C------------H
Scheme 5: The attempts at esterification of Compound II and
Compound III using Fischer esterification
22


2.2.1 Attempted Fischer Esterification of Compound II
(5)
The Fischer esterification reaction had been attempted Carolina Wilson
without success (1). She did not have time to fully confirm that this reaction would
not work. Futhermore, she had tested only a single batch of Compound n. Our first
goal therefore was confirm that this reaction does not work using a variety of standard
Fischer esterification conditions. Wilsons original experiment was repeated 2 times
using new batches of Compound II, but without success. Compound II was suspended
in anhydrous methanol. HC1 gas was passed through this mixture for 30 minutes and
the reaction flask was stoppered. The suspension was stirred overnight. At first the
suspended solid dissolved in the acidic solution, but as bubbling of the HC1
continued, it reprecipitated. During the work up a white precipitate was recovered by
filtration and a yellow residue was obtained by evaporating the filtrate.
H-NMR analysis of the white precipitate dissolved in DaO/NaOD solution
showed no singlet at 3.8 ppm (CH3-O-). Resonances at 1.18, 1.24, 1.72 and 1.78 ppm
correspond to the original Compound II.
The 'H-NMR spectrum (D2O) of the yellow residue is shown in Fig 2.2.1. No
singlet at 3.8 ppm (CH3-O-) was obtained. Unexpected singlets at 3.66, 3.5 and
3.1 ppm were
23




obtained. The apparent doubling of these is an artifact of the instruments tuning. The
overlapping resonances at 2.2-2.0 ppm peak correspond to the starting material. The
'H-NMR results, indicating no ester peak at 3.8 ppm, confirmed that standard the
Fischer esterification method does not work well on compound II. We stopped
pursuing this reaction at this point.
25


2.2.2 Attempted Fischer Esterification of Compound III
(5)
The amino group of Compound HI is protected by a formyl group, which
should allow this compound to dissolve more easily. Compound III was suspended in
an excess of anhydrous methanol. When HC1 gas was passed through for a few
minutes, all dissolved. As more hydrogen chloride gas was passed into the clear
solution, a white suspension was formed. The suspension was stirred at room
temperature overnight. During the work-up the white precipitate was recovered by
filtration and the filtrate was evaporated to obtain a white residue.
The 'H-NMR spectrum (d-methanol) of the white residue is shown in Figure
2.2.2. No methyl ester peak at 3.8 ppm was obtained. Peaks considered to be the d-
methanol (3.29, 4.87 ppm) and cyclohexane ring (2.29-2.19 ppm) were identified.
The identity of the singlet at 3.9 ppm is unknown.
The formyl proton peak has disappeared from the 'H-NMR spectrum of the
reaction product. Absence of the formyl proton peak is attributed to the acidic
conditions. In acidic conditions the formyl group is apparently cleaved before the
formation of ester can occur. It had been hoped that esterification would be fast
enough to allow formation of the diester. But apparently the formyl group was
cleaved too fast and the molecule reverted to Compound 13, and precipitated. We
26


stopped investigations of Fischer esterifications at this point. The next steps examine
new methods to make an ester.
27


0
to
00
Fig 2.2.2
'H-NMR spectrum in d-methanol at 200 MHz of the residue from the
attempted Fischer esterification of Compound in


2.3 Attempted Esterification with Methyl Iodide
The second proposed pathway for esterification was methylation: reaction of
the carboxylate salt with methyl iodide. The attempted esterification of Compound HI
using CH3I was performed following a general procedure by Gamer and Park (6).
This same type of esterification was carried out in our laboratory using a different
carboxylic acid, with good results and high yield. Therefore we tried this method with
Compound HI. The first step of the reaction is to dissolve the starting amino-protected
material using DMF or DMSO. But Compound HI only partially dissolved in these
solvents. The second step is deprotonation of the COOH group using an inorganic
base, K2CO3, or an organic base, Et3N. The third step is nucleophilic attack of the
carboxylate ion on the methyl group of methyl iodide, supposedly to yield the methyl
ester. The different reaction conditions used are shown in Scheme 6.
29


o
o
HC =0
HC =0
1. DMSO
2. K2C03
3. CH3I
O
HC =0
Scheme 6: The attempts at esterification of Compound HI
using methyl iodide
30


2.3.1 Attempted Methylation of Compound III in DMF
using K2CO3 as the Base
Using DMF as the solvent the reaction was carried out 2 times, with no
evidence of formation of the desired dimethyl ester compound. Compound in was
suspended in DMF and K2CO3 was added. Methyl iodide was added to this reaction
mixture dropwise, followed by stirring at room temperature for 1 hour. After the work
up process, a residue was obtained.
The residue was analyzed by 'H NMR (CDCI3). No methyl ester peak was
obtained. Only signals at 8.1, 2.9 and 3.05 ppm (DMF) and at 4.1 ppm, 2.0 ppm, and
1.3 ppm (EtOAc) were observed. No formyl peak or cyclohexane resonances were
present.
This reaction apparently failed because Compound IH does not fully dissolve
in DMF. The salt is probably much less soluble. Failure to detect any of Compound II
or its derivatives in the organic extract of the reaction mixture indicates that it was
removed during filtration of the reaction mixture. To help this reaction we needed to
change the solvent. Solubility tests were carried out and the best prospect was
DMSO.
31


2.3.2 Attempted Methylation of Compound III in DMSO
using K2CO3 as the Base
The esterification reaction was carried out two times using the same
procedure, described above, except that the solvent was DMSO. Compound m was
suspended in DMSO and K2CO3 was added. Methyl iodide was added dropwise to
this reaction mixture. After the work up process, a white precipitate and a residue
were obtained. *H NMR of the residue showed no methyl ester peak at 3.8 ppm. At
this point, a new way was sought to prepare the carboxylate anion in solution. The
inorganic base was changed to an organic base, Et3N, in hopes of making the dianion
more soluble.
2.3.3 Attempted Methylation of Compound III in
DMSO using an Organic Base, Triethylamine
The Compound EH was dissolved with stirring and gentle heating in DMSO.
Triethylamine was added to this mixture and it was stirred at room temperature for 3
hours. TLC analysis of the reaction mixture before work up gave no indication of a
new, faster moving component. During the work up by extraction, three layers were
obtained:an upper organic layer, a middle (yellow precipitate) layer, and an lower
water layer. H NMR spectra of the contents of the organic and middle layers showed
32


no methyl ester peak at 3.8 ppm. Further attempts to apply this method of
esterification were abandoned this point.
33


2.4 Third Proposed Pathway to Compound III:
Esterification using DCC as a Coupling Reagent
DCC is used for coupling steps of peptide synthesis and is also used for the
preparation of esters. DCC is a highly reactive activator of carboxyl groups, and
gives high yields within a short time. The three different attempted esterification
reactions using Compound HI with DCC as a coupling reagent are shown in
Scheme 7. Attempted esterifications of Compound EH using DCC as a coupling
reagent were carried out following the general procedure Bodanzsky and
Bodanszky (7). The first step involves addition of the N-protected amino acid to the
reagent (DCC) to form a reactive intermediate, an O-acylisourea. Such
intermediates have never been isolated. In the second step, direct nucleophilic
attack of the alcohol on the reactive intermediate (O-acylisourea) yields formation
of an ester bond. Also formed is the by-product N, N-dicyclohexylurea (DCU),
which is not very soluble and can be removed by filtration. In these reactions both
methyl alcohol and para-nitrophenol were used. No formation of an ester was
demonstrated in any of these reactions.
34


H-CO-NH
CO-O-
NH-CO-H
-N02
H-CO-NH
CO-O-CH3
NH-CO-H
HOOC NH-CO-H
At 70 C
><
1. DMSO
2. DCC
3. HO-f ^ -N02
>-o
H-CO-NH C0-0-
-NO,
NH-CO-H
Scheme 7: Proposed esterifications of the carboxyl group of Compound in
by DCC
35


2.4.1 Attempted Esterification of Compound III
using DCC and Methanol in DMSO
Compound m was dissolved DMSO with warming, then DCC was added.
The reaction mixture was treated with an excess of methanol. The reaction mixture
was stirred at room temperature for three hours. After a work-up process by organic
extraction a residue was obtained.
The residue was analyzed by 'll NMR spectroscopy (d6-acetone). But no
methyl ester peak at 3.8 ppm was present. Resonances corresponding to dicyclohexyl
urea at 3.5 and 1.25-1.35 ppm, and to the ring of Compound IH at 1.7-1.6 ppm were
observed.
The organic and water extraction layers were also analyzed by TLC in System
A. h positive spots with Rf values of 0.46 and 0.42 were obtained. These Rf values
correspond to the starting materials. There was no indication of the formation of a
methyl ester.
36


2.4.2. Attempted Esterification of Compound III
using DCC and Para-nitrophenol in DMSO
In this reaction the hydroxyl component was changed. Bodanszky and
Bodanszky have used DCC to prepare para-nitrophenyl esters routingly, and with
high yields (7).
The same procedure was carried out as above, except para-nitrophenol was
used. Compound HI was dissolved in DMSO, and DCC was added. Para-nitrophenol
was added to the reaction mixture, and stirred for two days. After the work-up a
residue was obtained. The residue was dissolved in de-acetone for XH NMR analysis
(Fig 2.4.1). The expected chemical shifts were 7.3 ppm and 8.3 ppm (a,b quartet,
para-substituted benzene ring), 8.1 and 7.9 ppm (s, formyl proton peaks) and 1.6-1.8
ppm (m, cyclohexane ring peaks). Peaks corresponding to these expected signals
were observed, but the integrals were not in the correct proportions. The broad signals
at 3.3 ppm and 1.0-1.4 ppm did not correspond to any expected product. The 'll
NMR result was ambigious. Therefore this reaction was tried at 70 C, to force
completion.
37




2.4.3 Using DCC as a Coupling Reagent
(Para-nitrophenol at 70 C)
To force the reaction to completion, the DCC coupling was carried out
reaction at 70 C. All of the previous procedure was repeated, except the temperature
was kept at 70 C for three hours. After the work-up the residue was analyzed by 'H
NMR spectroscopy (d6-acetone).
The spectrum showed a broad peak between 3.6-4.7 ppm which could not be
assigned. At 2.05 ppm is solvent peak (de-acetone). At 1.2-1.9 ppm were signals
corresponding to the cyclohexane ring protons. There were no peaks in the araomatic
region.
Analysis by TLC showed no evidence of a more mobile component, which
could be expected if an ester had been formed. With no positive evidence for ester
formation, we stopped investigation of DCC-mediated reactions at this point.
i
i
39


2.5 Attempted Esterifications Using SOCl2
The fourth proposed pathway for esterification was using SOCl2 to
activate the carboxyl group. We tried this method on both Compound II and
Compound in. The reaction conditions are shown in Scheme 8.
1 ,CH3OH (excess)
2. HC1 (g)
3. SOCl2

1. DM SO
2. SOCl2
3. CH3OH
O
CH3-O-OC NH-C--------H
Scheme 8: The attempts at esterification of Compound II and
Compound III using SOCl2
40


2.5.1 Attempted Esterification of Compound II in
Methanol using Thionyl Chloride (8)
Compound II was suspended in anhydrous methanol. HC1 gas was passed
through the white suspension for 5 min to obtain a clear solution. SOCI2 was added,
then reaction mixture was refluxed. After evaporation of the solvent, a white residue
was obtained. A *H NMR spectrum (D20/Na0D) of the white residue is shown in Fig
2.5.1. No evidence at 3.8 ppm of a methyl ester peak was obtained. The singlet at 4.8
ppm corresponds to the solvent D2O. The medium resonances at 1.86, 1.64, 1.15 and
1.11 ppm correspond to the starting material. The source of the singlet at 3.0 ppm
peak is unknown. Lack of mobility of the product on TLC indicates that an ester is
probably not present.
41


-p-
K>


2.5.2 Attempted Estarification of Compound III using
Thionyl Chloride in DMSO
Compound IE was suspended in DMSO, then thionyl chloride was added.
The suspension was refluxed for 1 hour. Methanol was added to the cooled reaction
mixture. The reaction mixture was refluxed again. After the work-up, an oily residue
was obtained. The residue was dissolved in de-acetone and then in d-methanol for 1H
NMR analysis. Not all of the peaks observed are considered to be solvent peaks
(DMSO, d-acetone and d-methanol). The filtrate and white precipitate were analyzed
by TLC in 4:1:1 (butanol: acetic acid: water), to compare them with the starting
material. Ninhydrin and I2 positive spots with Rf value of 0.22 and 0.34 were
observed in the white precipitate and the filtrate, respectively. The latter corresponds
to the starting material. The former, with a lower Rf, does not correspond to the
expected mobility of the ester.
43


2.6. Attempted Methanolysis of the Nitrile Functions
of Compound I
The fifth proposed pathway for esterification used a different starting material,
Compound I. The intention was to convert the two nitrile functions directly to methyl
esters using either acid or base catalysis (9), as shown previous in Scheme 2 (p.9).
2.6.1 Attempted Methanolysis of Compound I
in Acidic Conditions
Compound I was dissolved in methanol and dry HC1 gas added. The resulting
reaction mixture was refluxed and produced a white precipitate. The suspended solid
was then collected by filtration, dried and weighed. (recovery:75%)
This residue was analyzed by 'H-NMR spectroscopy (D2O) and TLC. The
pair of doublets between 2.0and 2.6 ppm and the Rf value on TLC were the same as
observed for the starting material.
The 25% of the material that was recovered from the filtrate was analyzed by
H-NMR spectroscopy. It partly dissolved in d-methanol (Fig 2.6.1). No clear singlet
ester peak at 3.8 ppm was observed. The singlets seen at 3.31 and 4.92 ppm
correspond to d-methanol. The origins of the signals at 3.86 and 3.67 ppm and 2.38,
2.12, 1.84 ppm are unknown. The residue was analyzed by TLC analysis in both
System A and System B. A UV and ninhydrin positive spot at the origin was obtained
44


with System B. This Rf value is very close to that of the starting material and does not
indicate conversion of that compound to an ester.
45


Fig 2.6.1 'H-NMR spectrum in d-methanol at 200 MHz of the product from treating
Compound I with methanol in acidic conditions


2.6.2 Attempted Methanolysis of Compound I in
Basic Conditions
Compound I was dissolved in methanol, and an equilent amount of sodium
methoxide was added to this solution. The mixture was refluxed for 2.5 hours. After
cooling, 1M HC1 was added to this reaction mixture, and it was stirred overnight to
complete hydrolysis. A white precipitate and a filtrate were obtained.
The white precipitate constituted 22% of the mass of the starting material. Its
ll NMR spectrum (D2O) showed only doublets at 2.4 and 2.1 ppm, corresponding to
the starting material.
The filtrate was extracted and the yielded yellow crystals. The 'H NMR
spectrum of these crystals partly dissolved in c^-acetone, (Fig 2.6.2) shows a complex
mass of peaks between 1.6 and 2.6 ppm. Only the singlet at 2.6 ppm, corresponding
to dfi-acetone, could be assigned with confidence. The origin of the signals at 3.2 and
3.6 ppm are unknown, but neither corresponds to the desired methyl ester peak, which
should be at 3.8 ppm. The aqueous and organic layers from above were analyzed by
TLC in System A. An I2 and nynhydrin positive spot of Rf value of 0.4 was obtained
from the water layer. The organic layer contained only a UV positive spot at the
origin. Further attempts to recover an ester by extraction of the remaining aqueous
phase at pH 10 proved futile.
47




2.7 Attempts to Cyclize Compound II by Heating
The sixth proposed pathway to the bicyclic amide avoids esterification. It is a
thermal condensation reaction of Compound n. This approach was based on a report
by Houben and Pfau (10), in which 4-amino-1-cyclohexanecarboxylic acid was
cyclized by heating. The corresponding conversion for Compound II is shown in
Scheme 3 (p.10).
2.7.1 Attempt to Cyclize Compound II by
Heating with a Torch
A sample of a few mg of Compound II in test tube was heated briefly over a
propane torch. Water condensation occurred in the upper portion of the test tube. The
color changed to brown. This brown residue was added to water and was analyzed by
TLC and *H-NMR spectroscopy to compare it to the starting material.
The *H-NMR spectrum of the portion of the residue that was soluble in D2O
correspond to starting material. The Rf value of the soluble product was also same as
the starting material. It is apparent that no altered (cyclized) material dissolved in the
added water. The next experiment was an attempt to control the changes in
Compound II brought about by heating.
49


2.7.2 Attempt to Cyclize Compound II by
Heating in a Heating Block
An attempt to cyclize Compound II was carried out using a heating block.
Compound II in an evacuated tube was heated overnight at 150 C. Water
condensation in the upper portion of the reaction tube was observed after even just
one hour of reaction. A white solid was obtained.
A H-NMR spectrum of a D2O extract of this white precipitate showed no
cyclohexane peaks. The white precipitate and D2O / DC1 were mixed in an NMR tube
and left overnight. In the morning the test tube was found broken, possibly from
expansion of the solid. The residual solvent was found to be at pHl.
Compound II and the simple cyclization product should both soluble in
D2O/DCI in acidic conditions, but the heat-altered sample did not dissolve in these
solvents. This behavior indicates that the residue was not compound II and probably
not the simple cyclization product. It may have been polymeric. These studies will
need repeating at several temperatures, with more extensive analysis.
50


2.8 Conclusions
The synthesis of the formylated bis-amino acid (Compound El) was studied.
The procedure was optimized by formylation in different concentrations of formic
acid. Using 96% formic acid and 4% water the yield of Compound III was 87-90%.
In 99 % formic acid yields were much poorer.
The elemental analyses of Compound I and Compound II were obtained. The
values were very close to the theoretical values. Compound II precipitates with two
molecules of water per organic unit.
Synthesis of the bis-amino acid ester was attempted using several approaches.
Standard Fischer esterification apparently does not work for reasons of poor
solubility. Using SOCI2 to activate the carboxyl functions under similar conditions
also failed.
Methylation of the carboxylate group by methyl iodide, attempted with different
solvent systems (DMF or DMSO) and different bases (K2CO3 or Et3N ) did not work.
Solubility in DMF appears to be too limited. Other bases in conjunction with DMSO
might be explored.
Attempted esterifications using DCC activation and different alcoholic
conditions were unsuccessful. DCC reacted to form a white precipitate (presumably
DCU). In future studies the use of catalysts with DCC (for example dimethylamino
pyridine) or other peptide coupling reagents could be tried.
51


An attempt was made to use different starting material, 1,4-diamino-1,4-
dicyanocyclohexane, to make an ester The compound was treated with methanol and
HC1. It was also treated with sodium methoxide. Neither set of conditions yielded an
identifiable ester.
Direct heating of the bis-amino acid to cause a cyclic condensation reaction
gave ambiguous result. Water was condensed and the compound underwent
irreversible changes. There was no direct evidence of the formation of the desired
bicycle-amide, however. A through study varying conditions of temperature and time
will be needed to determine if this approach to the bicyclic molecule can be made
practicable.
52


3. Experimental
3.1 Reagents
1,4-Cyclohexanedione (98%), formic acid (96%), DCC (dicyclohexylcarbodiimide)
99%, benzyl bromide (98%), and DMF (dimethylformamide 99.9%) were purchased
from Aldrich. Methyl iodide (99.8%), acetic anhydride (99%), glacial acetic acid
(99.9%), and phosporus pentoxide (99.8%) were purchased from Fischer Scientific.
Triethylamine (99%), thionyl chloride (99.5%), and sodium cyanide (95%) were
purchased from Acros Organics. Ethyl acetate (99%), dimethylsulfoxide (99.9%),
hydrochloric acid (37%), ammonium chloride (99%), and anhydrous magnesium
sulfate were purchased from Mallinkrodt.
3.2 General Methods
3.2.1 Thin Layer Chromatography
Thin layer chromatography (TLC) was performed using aluminum plates
precoated with 0.2 mm layer of silica gel (F254) (EM science). Samples were spotted
on the plates using a lpL micropipette and the chromatograms were developed in
mixed solvent systems, either butanol:acetic acid:water (4:1:1), System A, or
chloroform:methanol (9:1), System B. The identification of the components was
determined by one of the following methods:
A: The plate was observed under ultraviolet light (254 nm)
53


B: The plate was immersed in silica gel saturated with iodine for a brief period.
C: The plate was sprayed with a solution containing 2% ninhydrin in acetone and then
heated for few minutes at under heater gun.
Under one or more of the previous conditions components were seen as dark spots on
a lighter background.
3.2.2 Nuclear Magnetic Resonance Spectroscopy
Nuclear magnetic resonance analyses were performed using a Varian 200
MHz spectrometer. Chemical shifts, 5 are reported in ppm from TMS (6=0.00).
Solutions were prepared in CDCI3, D2O, D2O with DC1 or NaOD, d^-acetone or, d-
methanol.
3.2.3 Infrared Spectroscopy
IR spectra was recorded on 360 FT-IR spectrometer. Spectra were obtained at
room temperature. Solid samples were placed under a concave cover on a flat
diamond window and recorded directly.
54


3.3 Experimental Procedures
3.3.1 Synthesis of l,4-Dicyano-l,4-diamino
cyclohexane (Compound I)
Compound I (l,4-dicyano-l,4-diaminocyclohexane) and Compound II ( 1,4
diaminocyclohexane-l,4-dicarboxylic acid) were synthesized using the methods of
Carolina Wilson (1).
The starting material l,4-cyclohexanedione( 7.46 g,0.066 mole) was dissolved
in 90 ml of a 50% solution of aqueous methanol. Ammonium chloride (7.08g, 0.13
mole) was then added to this solution. Sodium cyanide (6.48g, 0.13 mole ) was added
to the reaction mixture and stirred at room temperature for 48 hours. The precipitate
that was formed. It was collected by filtration, dried, and weighed. The product was
analyzed by NMR, IR, and TLC. 'H-NMR (D20/DC1): 8 (ppm) 2.33-2.28 (d,
hydrogens of cyclohexane ring): 1.91-1.86 (d, hydrogens of cyclohexane ring)
Compound I was dissolved in the acidic solution for TLC analysis in Systems
A and B. An iodine, ninhydrin positive spot with Rf value of 0.41 was obtained in
System A. A single UV, iodine, and ninhydrin positive spot at the origin was obtained
in System B. These correspond to the spectrum and TLC data obtained by Wilson.
Elemental analysis and an IR spectrum were performed on Compound I.
55


3.3.2 Synthesis of 1,4-Diamino-l,4-cyclohexane
Dicarboxylic Acid (Compound II)
Compound I was suspended in 12 M hydrochloric acid solution. The
reaction mixture was then stirred at room temperature for 48 hours. After the stirring
period, the white suspension was diluted with 30 ml of water and heated under reflux
for another 48 hours. After the work-up process, Compound II was obtained. 'H-
NMR (D2O/DCI): 8 (ppm) 1.86-2.03 (m, hydrogens of cyclohexane ring; 'H-NMR
(D20/Na0D): 8 (ppm) 1.90-1.85 (d, hydrogens of cyclohexane ring): 1.35-1.31 (d,
hydrogens of cyclohexane ring)
56


3.3.3 Synthesis of 1,4-Diformylamino of 1,4-cyclohexane
Dicarboxylic Acid (Compound III)
Compound II (0.465 g, 2.3 mmol) was added to 25 mL of 96% formic acid.
The reaction mixture was heated in a hot water bath (50-55 C) and stirred. When
Compound II had dissolved completely in the formic acid, it formed clear yellow
solution. To this solution, 5 mL of acetic anhydride was added dropwise. After 10
minutes a white precipitate was observed. The resulting suspension was stirred at
room temperature for 2 hours, at the end of which 5 mL of ice water was added to
remove any excess acetic anhydride. The final reaction mixture was a white
suspension. The white precipitate was collected by filtration (sintered glass funnel),
washed with water, and dried. A yield of 0.4 lg of product (88%) was obtained.
Compound HI was dissolved in FLO/NaOH for TLC analysis in System A and B. A
single UV and h positive spot at the origin was obtained in System B. A single UV
and L positive spot with an Rf value of 0.35 was obtained in System A.
*H-NMR (D20/NaOD): 6 (ppm) 8.08 (s, formyl group); 7.97 (s, formyl group), 1.79-
2.00 (m, 8H, cyclohexane ring)
IR: 3362 cm'1 (NH strech), 2888 cm'1 (formyl CH stretch), 1643 cm'1 (formyl CO
stretch and carboxylic acid CO stretch)
57


3.3.4 Attempted Synthesis of 1,4-dimethyl-l,4-diamino
Cyclohexane Dicarboxylate (Compound IV )using
Fischer Esterification
Compound II (2.0g, 9.9 mmole) was suspended in anhydrous methanol (250
mL). The suspension was placed in an ice bath and dry hydrogen chloride was passed
into it. After 25 minutes a clear solution was obtained. Further addition of hydrogen
chloride gas (15 min) transformed the clear solution back to a suspension that was
then sealed and stirred overnight at room temperature. After the stirring period, the
white suspension was collected by filtration, dried and weighed. Yield: 0.038g (20%).
H-NMR analysis of the white precipitate dissolved in D20/Na0D solution showed
resonances at 1.18, 1.24, 1.72 and 1.78 ppm (corresponding to Compound II). The
cloudy filtrate was allowed to stand for 24 hours. After 24 hour standing the
supernatant was decanted, yielding another white precipitate and a clear solution. The
latter was taken to dryness to obtain a white- yellow residue which was dissolved in
D2O for 'H-NMR analysis. Peaks at 3.66, 3.5, 3.1 and 2.2-20 ppm were observed.
The residue was redissolved 15 mL water, and then Na2CC>3 was added to raise pH
value to pH 12. The aqueous solution was extracted with ethyl acetate (3x30 mL).
The combined organic layer was taken to dryness by oil pump to obtain a yellow
liquid. The yellow liquid was dissolved in D2O for 'H-NMR analysis. Signals at 1.98,
1.92, 1.68 and 1.64 ppm, (corresponding to the starting material) and at 3.6 and 3.1
ppm were obtained.
58


3.3.5 Attempted Synthesis of Dimethyl- 1,4-diformylamino-
1,4-cyclohexane Dicar boxy late (Compound V)
Using Fischer Esterificaion
Compound HI (0.105g, 0.41 mmole) was suspended in anhydrous methanol
(20 mL). The suspension was placed in an ice bath and dry hydrogen chloride was
passed into it for 30 minutes. After 10 minutes the suspension became a clear
solution. The reaction mixture was sealed and stirred at room temperature overnight.
After the stirring period, a white suspension had formed. It was collected by filtration,
giving a clear filtrate solution. The filtrate was taken to dryness to obtain a white
residue. 'H-NMR analysis of the white residue dissolved in d-methanol showed peaks
attributed to d-methanol (3.1 and 4.8 ppm) and cyclohexane ring peaks (2.32-2.21
ppm). The white residue was analyzed by TLC in Systems A and B. A UV, I2 and
ninhydrin positive spot with Rf value of 0.34 was seen in System A. In System B the
UV, h and ninhydrin positive spot remained at the origin.
59


3.3.6 Attempted Synthesis of Compound V using
Methyl Iodide and K2CO3 in DMF
Compound III (0.2g, 0.77 mmole) was suspended in 1.8 mL of DMF. Some
dissolved and some remained as a white solid on the bottom of the vial. Potassium
carbonate (K2CO3) was added to the reaction mixture, which was placed in an ice
water bath for 10 min. Methyl iodide (3.8 mL, 61 mmole) was added dropwise to the
white suspension. The reaction mixture was warmed to room temperature and stirred
for one hour. It was then filtered by suction and the filtrate was partitioned between
ethyl acetate (25 mL) and water (25 mL). The organic phase was washed with brine,
dried over magnesium sulfate, filtered and concentrated to give a yellow oily residue.
The residue were analyzed XH NMR spectra in CDCI3. All of the peaks detected are
considered to represent DMF and EtOAc.
60


3.3.7 Attempted Synthesis of Compound V using
Methyl Iodide and K2CO3 in DMSO
Compound HI (0.2g, 0.77 mmole) was suspended 1.8 mL DMSO with
stirring. The suspension was placed hot water bath and some of it dissolved. To the
reaction mixture was added 0.3g K2CO3. The vial was placed in an ice water bath for
10 min. Methyl iodide (3.8 mL, 61 mmole) was added dropwise to this cooled
mixture. The reaction mixture was stirred at room temperature overnight. A white
suspension was obtained. This suspension was filtered and the filtrate was partitioned
between ethyl acetate and water. The organic layer was washed with brine, dried over
MgS04 and taken to dryness by rotary evaporation. The residue was characterized by
*H NMR spectroscopy (CDCI3). Only ethyl acetate and CDCI3 peaks were obtained.
The white precipitate from the reaction mixture was dissolved in D2O/DCI and a *H
NMR spectrum was taken. It looked like that of Compound IE.
61


3.3.8 Attempted Synthesis of Compound V using
Methyl Iodide and Triethylamine in DMSO
Compound III (0.2g, 0.78 mmole) was dissolved with stirring and gentle
heating in 1.8 mL of dimethylsulfoxide (DMSO). To the mixture was then added 0.2
mL (1.44 mmole) of triethylamine, resulting in a white suspension. The white
suspension was placed in an ice water bath. After stirring for 10 minutes in an ice
water bath, methyl iodide (3.8 mL, 61 mmole) was added. The ice water bath was
removed and the reaction mixture was stirred at room temperature for 3 hours. The
white suspension turned to a brown clear solution, which was analyzed by TLC in
System A. An L and UV positive spot with Rf value of 0.43 was obtained. The brown
clear solution was partitioned between ethyl acetate and water. A lower water layer, a
middle (yellow precipitate) layer and an upper organic layer were obtained. The
organic layer was washed with brine, dried over magnesium sulfate, filtered and taken
to dryness by oil pump to obtain a residue. This residue was suspended in D2O for ]H
NMR analysis but a black precipitate was obtained. This mixture was evaporated
again and the residue was dissolved in d-methanol and then in d6-acetone for 'H
NMR analysis. All peaks observed were considered to be solvent peaks (DMSO,
CD3OD, EtOAc or d-acetone) except for a signal at 1.4-1.2 ppm, which corresponded
to the cyclohexane ring peaks.
62


The middle extraction layer (yellow precipitate) was dissolved in d-acetone for 'H
NMR analysis. Some of it did not dissolve. No signal at 3.8 ppm (CH3 -0-) was
observed.
63


3.3.9 Attempted Synthesis of Compound V in DMSO
using DCC as an Activating Agent
A solution of Compound IE (O.lg, 0.38 mmole) was prepared in 0.9 mL of
DMSO, and 0.198g (0.96 mmole)of DCC in 1.0 mL of DMSO was added. The
solution was then treated with 0.1 mL (2.5 mmole) of an anhydrous methyl alcohol.
After 5 minutes a suspension appeared in the solution. The reaction mixture was
stirred at room temperature for 3 hours. At the end of the reaction 0.05 mL (0.84
mmole) of 97% acetic acid was added to the solution. The precipitated
dicyclohexylurea was removed by filtration and washed with cold methanol. Yield
0.12g. The filtrate was partitioned between water (30mL) and ethyl acetate (30mL).
The organic phase was a white suspension. The organic layer was filtered and a clear
organic layer and a white precipitate were obtained. The clear organic layer was dried
over magnesium sulfate, filtered and concentrated to give white residue. The white
residue was dissolved in cL-acetone for *H NMR analysis. Some of it dissolved but
some did not. No signal at 3.8 ppm (CH3-O-) was observed in the NMR spectrum of
this product. The organic and water layer were analyzed by TLC in System A. I2
positive spots with Rf values of 0.46 and 0.42 were obtained. The water layer was
taken to dryness to obtain a residue which was dissolved D2O for 'H NMR analysis.
No signal at 3.8 ppm was observed in the spectrum of this product, either.
64


3.3.10 Attempted Synthesis of Bis-nitrophenyl-1,4-
diformylamino-1,4-cyclohexane dicarboxylate
using DCC as an Activating Agent
A solution of 0.1 g (0.38 mmole) of Compound HI in 0.9 mL of DMSO was
prepared with gentle heating and stirring, and 0.178g (0.89 mmole) of DCC in 1.0 mL
of DMSO was added. The solution was then treated with 0.437g (3.1 mmole) of
recrystallized para-nitrophenol (yellow needle). The reaction mixture was stirred at
room temperature. After stirring 48 hours the precipitated urea was removed by
filtration and the filtrate was partitioned between ethyl acetate (30 mL) and 5%
sodium carbonate (30 mL).The organic layer was washed with 5% Na2C03 (7x10
mL) to extract the unreacted para-nitrophenol and then with brine (40 mL). The
organic layer was dried over MgS04, filtered and taken to dryness to obtain a residue
which was taken up in CDCI3 and d^-acetone, sequentially, for 'H NMR analysis. 7.3
ppm (doublet), 8.3 ppm (doublet )both from the benzene ring, and 8.1 and 7.9 ppm
(formyl proton peaks ) and ppm (cyclohexane ring peaks). The integrals were not in
the expected ratios. The remaining residue was passed through a silica gel column
using acetone as the eluent. The eluant was evaporated. The residue was dissolved d-
acetone for 'H NMR analysis. No ester peak or cyclohexane peaks were observed.
65


3.3.11 Second Attempted Synthesis of Bis-para-
nitrophenyl-l,4-diformylamino-l,4-
cyclohexanedicarboxylate using DCC as an
Activating Agent at Elevated Temperature
A solution of O.lg (0.38 mmole) of Compound IE was dissolved with gentle
heating and stirring in 0.9 mL of DMSO and 0.18g DCC in 1.0 mL of DMSO was
added. The solution was then treated with 0.437g (3.1 mmole) of recrystallized para-
nitrophenol (yellow needles). The reaction mixture was placed in hot water bath and
stirred at 70 C for 3 hours. After stirring period hot water bath was removed and the
reaction mixture allowed to cool. The precipitated urea was removed by filtration and
the filtrate was partitioned between ethyl acetate (30 mL) and 5% sodium carbonate
(30 mL). The organic layer was washed with 5% Na2CC>3 (7x10 mL) to extract the
para-nitrophenol and then washed with brine (40 mL). The organic layer was then
dried over MgSCL, filtered and taken to dryness to obtain a residue which was
dissolved in cL-acetone for 'H NMR analysis. The spectrum showed a broad peak
between 3.6-4.7 ppm which could not be assigned. At 2.05 ppm is solvent peak (cL-
acetone). At 1.2-1.9 ppm were signels corresponding to the cyclohexane ring protons.
There were no peaks in the aromatic region. The organic and water layers from the
extraction were also analyzed by TLC in System B. Analysis by TLC showed no
evidence of a more mobile component, which could be expected if an ester had been
formed.
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3.3.12 Attempted Synthesis of Compound IV using
SOCI2 in Methanol
Compound II (O.lg, 0.49 mmol) was suspended in anhydrous methanol (40
mL). Dry hydrogen chloride was passed through the white suspension for 5 minutes
to obtain a clear solution. The flow of hydrogen chloride was then shut down. To the
clear solution was added 0.2 mL (2.7 mmole) of SOCI2, dropwise with effervescense.
The reaction mixture was refluxed for 1 h, resulting in a white suspension, which was
filtered. The filtrate was taken to dryness by oil pump to obtain a white residue. The
white residue only partially dissolved in D20/NaOD. A 'H-NMR spectrum of the
dissolved materials showed no evidence of a methyl ester peak. All peaks are
considered to be solvent (D20) and cyclohexane ring peaks (1.69, 1.64, 1.15 and 1.11
ppm). The white residue was analyzed by TLC in Sytem B. A UV, I2 and ninhydrin
positive spot at the origin was obtained.
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3.3.13 Attempted Synthesis of Compound V using
SOCl2 in DMSO
Compound III (O.lg, 0.38 mmole) was suspended in 1.0 mL DMSO and 0.26
mL (3.6 mmole) SOCI2 was added. The reaction mixture was refluxed for 1 hour. To
the cooled reaction mixture was added 0.8 mL of anhydrous methanol. The reaction
mixture was again refluxed for 1 hour. The reaction mixture was filtered and the
precipitate was removed by filtration and washed with methanol. The filtrate and
wash were combined and taken to dryness by under vacuum to obtain an oily residue.
The residue was dissolved in cL-acetone and in d-methanol for 'H-NMR analysis. Not
all of the residue dissolved. No singlet at 3.8 ppm (CH3-O-) was obtained. All of the
peaks observed are considered to be solvent peaks (DMSO, d-acetone and d-
methanol). The filtrate and white precipitate were analyzed by TLC in System A to
compare with the starting material. Ninhydrin and I2 positive spots with Rf value of
j0~22 .and 0.34 were observed in the white precipitate and the filtrate, respectively. The
latter corresponds to the starting material. The former, with a lower Rf, does not
correspond to the expected mobility of the ester.
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3.3.14 Attempted Synthesis of Compound IV by
Methanolysis of Compound I in Acidic Conditions
Compound I (0.114 g, 0.695 mmol) was dissolved in anhydrous methanol (40
mL). Dry hydrogen chloride gas was passed through the clear solution. After 5
minutes a white precipitate appeared. HC1 gas was passed through continuously 25
minutes and the white precipitate remained. The reaction mixture was refluxed for 2
hours. The suspended white solid was then collected by filtration, dried and weighed.
Yield: 0.085g (75%). *H-NMR analysis of this precipitate dissolved in D2O solution
showed no singlet at 3.8 ppm (CH3-O-). Resonances at 2.68, 2.63, 2.26, and 2.21 ppm
corresponding to Compound I, were obtained.
The filtrate from the reaction mixture was taken to dryness to obtain a residue.
The residue was dissolved in d-methanol for ^-NMR analysis. The expected
characteristic chemical shift of 3.8 ppm (CH3-O-) was not observed. The residue was
analyzed by TLC analysis in both System A and System B. A UV and ninhydrin
positive spot at the origin was obtained with System B. A UV and ninhydrin positive
spot at the origin and an I2, and ninhydrin positive spot with an Rf value of 0.46 were
obtained with the System A.
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3.3.15 Attempted Synthesis of Compound IV by
Methanolysis of Compound I in Basic Conditions
The mixture of Compound I (0.2g, 1.2 mmole) and 45 mL of anhydrous
methanol containing 0.13lg (2.4 mmole) of sodium methoxide was refluxed for 4.5
hours. The cooled solution was acidified with 3.25 ml of 1 M HC1 and stirred
overnight to complete hydrolysis. The resulting white suspension was filtered by
suction to obtain a white precipitate and a filtrate.
The collected white precipitate was dried and weighed. Yield 0.043g (21.5%).
It was dissolved in D2O for *H-NMR analysis. No signal at 3.8 ppm (CH3-O-) was
observed. Resonances at 2.4 and 2.1 ppm, corresponding to the starting material were
seen.
The yellowish filtrate was raised to pH 9-10, using sodium carbonate. The
mixture was partitioned between ethyl acetate (80mL) and water (80 mL). The water
layer was further extracted with ethyl acetate (2 times). The combined organic layer
was washed with brine, dried over magnesium sulfate, filtered and concentrated to
give yellow crystals. The yellow crystals were dissolved in cL-acetone for H-NMR
analysis. The expected chemical shift at 3.8 ppm (CH3-O-) was not observed.
The aqueous and organic layers from above were analyzed by TLC in system
A. An I2 and ninhydrin positive spot of Rf value of 0.4 was obtained from the water
layer. The organic layer contained only a UV positive spot at the origin. The aqueous
70


water layer was brought to pH 10, using sodium carbonate, and ethyl acetate was
added to obtain two layers. The organic layer was dried over magnesium sulfate,
filtered and concentrated to give a yellow oil. The yellow oil was dissolved in d-
methanol for 'H-NMR analysis. No ester peak at 3.8 ppm (CH3-O-) was observed.
Water layer was taken to dryness to obtain residue. Methanol dissolved a portion of
the residue. The methanol extract was taken to dryness to obtain a new residue, which
was dissolved d-methanol for 'H-NMR analysis. No ester peak at 3.8 ppm (CH3-O-)
was observed. The methanol extract was analyzed by TLC in system A. An I2 and
ninhydrin positive spot, Rf value of 0.5, was obtained in system A.
71


3.3.16 A Heating Reaction of Compound II
Using a Torch
Compound 13 (0.13g, 0.64 mmole) in a test tube was heated briefly over a
propane torch. Water condensation occurred in the upper portion of the test tube and
the color changed to a tan brown. Water was added. The pH of the suspension was
almost pH 6. There was still brown residue. This suspension was compared to
Compound II which became a white suspension when water was added. The solution
from the brown residue solution was filtered and the filtrate was taken to dryness to
obtain a new residue. This residue was dissolved in D2O for H-NMR analysis.
Resonances at 2.26, 2.22, 1.98, and 1.94 ppm corresponded to Compound n. No other
product peaks were present. Compound II and the heated sample were analyzed by
TLC (System A). An I2 positive spot (Rf 0.26) was obtained. This Rf value
corresponds to starting material.
72


3.3.17 A Heating of Compound II Using a Heating Block
A Temp-blok Module Heater was used. Before the reaction was carried out,
the heating block was calibrated. The heating block temperature was adjusted to 150
C. Compound II (0.05g, 0.25 mmole) was placed in a thick-walled, sealable tube.
Before the reaction was carried out, this tube was evacuated, then nitrogen gas was
introduced. The tube was again evacuated and sealed, then placed in the heating
block. The compound was heated overnight at 150 C. Condensation appeared in the
upper portion of the tube within an hour. After about 18 h, the heating block tube was
cooled, then opened. The reaction product was suspended in D2O for 'H-NMR
analysis. Only the HOD peak was observed. Then D2O/DCI was added to the sample,
but did not dissolve. The tube was left overnight, it was found broken in the morning.
The remaining solvent was found to be at pH 1.
73


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4. du Vigneaud, V.; Dorfmann, R.; Loring, H. S. J. Biol. Chem. 1932, 98, 511.
5. Kenner, G. W.; Preston, J.; Sheppard, R. C. J. Am. Chem. Soc. 1965, 18, 6239.
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8. Uhle, F.C.; Harris, L. S. J. Am. Chem. Soc. 1956, 78, 381.
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Synthesis of 4-diamino-1,4-cyclohexanedicarboxylic acid, a New Polymerization Monomer, and Its Derivatives, Amino Acid and Peptide Synthesis. Peptide Synthesis, Bioi. Chem. Am. Chem. Soc. Organic Synthesis, The Practice of Peptide Synthesis, Am. Chem. Soc. Am. Chem. Soc. Chem. Ber.