Synthesis of Potential Haptens with Morphine Skeleton and Determination of Protonation Constants

Article abstract of DOI:10.3390/molecules25174009

Vaccination could be a promising alternative warfare against drug addiction and abuse. For this purpose, so-called haptens can be used. These molecules alone do not induce the activation of the immune system, this occurs only when they are attached to an immunogenic carrier protein. Hence obtaining a free amino or carboxylic group during the structural transformation is an important part of the synthesis. Namely, these groups can be used to form the requisite peptide bond between the hapten and the carrier protein. Focusing on this basic principle, six nor-morphine compounds were treated with ethyl acrylate and ethyl bromoacetate, while the prepared esters were hydrolyzed to obtain the N-carboxymethyl- and N-carboxyethyl-normorphine derivatives which are considered as potential haptens. The next step was the coupling phase with glycine ethyl ester, but the reactions did not work or the work-up process was not accomplishable. As an alternative route, the normorphine-compounds were N-alkylated with N-(chloroacetyl)glycine ethyl ester. These products were hydrolyzed in alkaline media and after the work-up process all of the derivatives contained the free carboxylic group of the glycine side chain. The acid-base properties of these molecules are characterized in detail. In the N-carboxyalkyl derivatives, the basicity of the amino and phenolate site is within an order of magnitude. In the glycine derivatives the basicity of the amino group is significantly decreased compared to the parent compounds (i.e., morphine, oxymorphone) because of the electron withdrawing amide group. The protonation state of the carboxylate group significantly influences the basicity of the amino group. All of the glycine ester and the glycine carboxylic acid derivatives are currently under biological tests.

Full text of DOI:10.3390/molecules25174009

molecules
Article
Synthesis of Potential Haptens with Morphine
Skeleton and Determination of Protonation Constants †
István Köteles 1,
*
, Károly Mazák 1 , Gergo˝ Tóth 1,2 , Boglárka Tu˝z 1 and Sándor Hosztafi 1
1
Department of Pharmaceutical Chemistry, Semmelweis University, Hogyes Endre u. 9., H-1092 Budapest,
Hungary; mazak.karoly@pharma.semmelweis-univ.hu (K.M.);
toth.gergo@pharma.semmelweis-univ.hu (G.T.); tuz.boglarka@gmail.com (B.T.);
hosztafi.sandor@pharma.semmelweis-univ.hu (S.H.)
Department of Plant Anatomy, Institute of Biology, Eötvös Loránd University, Pázmány Péter sétány 1/C,
H-1117 Budapest, Hungary
2
*
Correspondence: koteles.istvan@pharma.semmelweis-univ.hu
†
Presented in part at the 23rd International Electronic Conference on Synthetic Organic Chemistry,
15 November–15 December 2019. Available online: https://ecsoc-23.sciforum.net/.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Julio A. Seijas Vázquez
Received: 7 August 2020; Accepted: 1 September 2020; Published: 2 September 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Vaccination could be a promising alternative warfare against drug addiction and abuse.
For this purpose, so-called haptens can be used. These molecules alone do not induce the activation
of the immune system, this occurs only when they are attached to an immunogenic carrier protein.
Hence obtaining a free amino or carboxylic group during the structural transformation is an important
part of the synthesis. Namely, these groups can be used to form the requisite peptide bond
between the hapten and the carrier protein. Focusing on this basic principle, six nor-morphine
compounds were treated with ethyl acrylate and ethyl bromoacetate, while the prepared esters
were hydrolyzed to obtain the N-carboxymethyl- and N-carboxyethyl-normorphine derivatives
which are considered as potential haptens. The next step was the coupling phase with glycine
ethyl ester, but the reactions did not work or the work-up process was not accomplishable. As an
alternative route, the normorphine-compounds were N-alkylated with N-(chloroacetyl)glycine ethyl
ester. These products were hydrolyzed in alkaline media and after the work-up process all of the
derivatives contained the free carboxylic group of the glycine side chain. The acid-base properties
of these molecules are characterized in detail. In the N-carboxyalkyl derivatives, the basicity of the
amino and phenolate site is within an order of magnitude. In the glycine derivatives the basicity
of the amino group is significantly decreased compared to the parent compounds (i.e., morphine,
oxymorphone) because of the electron withdrawing amide group. The protonation state of the
carboxylate group significantly influences the basicity of the amino group. All of the glycine ester
and the glycine carboxylic acid derivatives are currently under biological tests.
Keywords: hapten; vaccine; immunotherapy; N-demethylation; nor-compounds; morphine skeleton;
acid-base properties; protonation state; microspeciation
1. Introduction
Drug abuse is a worldwide problem. Even today, one of the most popular illegal substances is
morphine and its derivative heroin, besides cocaine and marijuana. These drugs have a very serious
potential to cause damage not just to the individual but to the whole society as well [1,2].
To help the drug addicts—and now let us just focus on the opiate users—there are few currently
available clinical possibilities. The most common way is methadone therapy. Methadone is a potent
opioid analgesic with longer duration of action compared to heroin or morphine. It can be a substitute
Molecules 2020, 25, 4009; doi:10.3390/molecules25174009
www.mdpi.com/journal/molecules

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for heroin and after withdrawal, the physical signs develop slower and seem to be less severe than
those after heroin withdrawal. These properties are the basis for the detoxification of heroin addicts
and methadone maintenance therapy, a long-term medication assisted treatment for opiate addiction.
Theoretically this protocol works fine but in practice it can be easily spoiled. Namely, if the patient
uses the illegal substance during the therapy all of the results that have been achieved so far are just a
waste of time, money and energy. Not even to mention that the level of tolerance decreases as well and
this can cause the death of the patient too. It is also a problem that relapses are quite common after
the therapy.
For this problem an alternative solution can be vaccination against these drugs [3]. Drugs of
abuse are small molecules that typically do not induce an antibody response following administration.
To induce antibodies against these kinds of molecules, structural changes have to be made to obtain
so called “haptens”. The hapten must be coupled to immunogenic proteins, called “carriers”.
These connected derivatives are typically drug-linker adducts, in which the linker has a terminal
functional group (i.e., carboxylic acid or aliphatic amine) that forms a covalent bond with the carrier.
The efficacy of these conjugate vaccines depends on several factors including hapten design, coupling
strategy, hapten density, carrier protein selection, and vaccine adjuvant [4,5].
Different opportunities are possible to functionalize the morphine structure: (i) O-alkylation the
C3 phenolic hydroxyl group; (ii) esterification of the C6 hydroxyl group or oxime formation of the C6
carbonyl group; (iii) N-alkylation of the nor-compounds.
Spector and Parker reported the synthesis of the first morphine hapten which was a 3-O-alkylated
compound [6,7] (Figure 1). Morphine was converted to 3-O-carboxymethylmorphine by reaction of
the base with sodium chloroacetate in ethanol and the 3-O-carboxymethylmorphine was coupled to
bovine serum albumin (BSA) with 1-ethyl-3(dimethylaminopropyl)-carbodiimide. This conjugate has
immunogenic properties and it was suitable for the quantitative determination of morphine in serum
by radioimmunoassay. Rubinstein and Ullman also prepared a 3-O-carboxymethylmorphine-BSA
conjugate, and the free COOH group was activated by preparing the mixed anhydride with isobutyl
chloroformate [8].
Figure 1. C3-hapten synthesis.
Buechler prepared 3-O-carboxymethylmorphine by refluxing of morphine base with bromoacetic
acid in ethanol [9]. Heimann et al. converted morphine C3 potassium salt in ethanol and the solution
was treated with ethyl bromoacetate. 3-Ethoxycarbonylmethyl-morphine was hydrogenated in the
presence of Pd-C catalyst, and then 3-ethoxycarbonylmethyl-dihydromorphine [10] was hydrolyzed.
For a C6 position modification, Wainer et al. prepared morphine-6-hemisuccinate ester by
the reaction of morphine base with succinic anhydride [11–14] (Figure 2). This compound was
simultaneously synthesized by Simon et al. [15]. In 1974 Bonese et al. used BSA conjugated

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morphine-6-hemisuccinate for the first time to vaccinate heroin dependent Rhesus monkeys [16].
After immunization, heroin self-administration in monkeys was blocked, and the antibody induced
blockade was shown to be dose-dependent.
Figure 2. C6 ester-hapten synthesis.
Pravetoni et al. developed haptens for vaccination of oxycodone and dihydrocodeinone [17–19].
These ketones were functionalized in position C6 (Figure 3) and C8. The reactions of oxycodone and
dihydrocodeinone with carboxymethyl hydroxylamine led to C6 O-substituted oxime derivatives.
These haptens were conjugated with BSA.
Figure 3. C6 oxime-hapten synthesis.
Codeinone was also transformed to a C8 substituted hapten. In this case the addition of an SH-group
of thioglycolic acid took place on the double bond (Figure 4). The step of conjugation can be carried out
with BSA.
Figure 4. C8-hapten synthesis.
Because BSA and KLH (keyhole limpet hemocyanine) are not allowed in human vaccinology,
Anton and Leff used tetanus toxoid to conjugate morphine-6-hemisuccinate [20].
Another way to design morphine haptens is to attach a COOH group, via a CH2 spacer, on the
nitrogen atom of normorphinans and the carrier protein is conjugated to the nitrogen atom (Figure 5).
Schneider converted normorphine with sodium bromoacetate to N-carboxymethyl-normorphine [21].

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Figure 5. Bridge N-hapten synthesis.
Gintzler et al. developed a radioimmunoassay method for the simultaneous determination of
morphine and codeine. Normorphine was transformed with ethyl bromoacetate and the product
N-ethoxycarbonylmethyl-normorphine (morphinan-17-acetic acid-7,8-didehydro-4,5-epoxi-3,6-
dihydroxy-ethyl ester) was then hydrolyzed [22]. The product of the hydrolysis was coupled to BSA.
Findlay et al. used ethyl γ-bromobutyrate to N-alkylate normorphine and norcodeine in order to
utilize a longer spacer [23]. The process was similar to Gintzler’s method. Herndon et al. also reported
the synthesis of N-ethoxycarbonylmethyl-normorphine in the reaction of normorphine with ethyl
bromoacetate. The ester was purified by silica gel column chromatography and hydrolysis of the ester
resulted in N-carboxymethyl-normorphine [24].
Morris et al. prepared N-succinic-normorphine by carbodiimide coupling of normorphine with
succinic acid [25]. This compound however did not contain basic nitrogen, after coupling to BSA
yielded an immunoconjugate which was produced antisera after animal immunizations.
Stowe et al. in 2011 synthesized several haptens for the immunization of heroin. These molecules
were conjugated to carrier proteins to receive heroin-vaccines [26
was treated with N-Boc- -aminobutanal and sodium triacetoxyborohydride to accomplish reductive
amination. After removing the protecting group with trifluoroacetic acid (TFA) the free amino group
was coupled with -tritylmercaptopropionic acid N-hydroxysuccinimide active ester. Removing the
,27]. 3,6-Diacetylnormorphine
δ
β
trityl protecting group with TFA liberated the mercapto group (HerHap Figure 6) making it possible
for further conjugation.
Figure 6. HerHap.
Recently a new type of functionalization of the aromatic ring has been elaborated, exchanging the
phenolic hydroxyl group with an isostere amino group in order to connect function groups.
Bremer and Janda used 3-dezoxy-3-aminomorphine as the starting material for the synthesis
of the heroin hapten [28]. After acetylation in the C6 position, this compound was N-demethylated

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and then N-alkylated to obtain the N-(
δ-aminobutyl)-derivative. Because this compound contained
an amide group in the C3 position instead of an ester, it was more stable against hydrolysis (Figure 7).
It is a key factor of hapten development as the heroin-hapten is not stable: at pH = 7.4 and at room
temperature, the shelf half-life is only 97 h. Because of this, the immunoconjugate practically has to be
used right after the preparation.
Figure 7. 3-acetamido heroin hapten.
Li et al. reported the synthesis of similar hapten molecules and these were conjugated to tetanus
toxoid [29]. The PrOxyHap (C3 hapten) contains a C3 aminogroup acylated with β-SH-propionic acid
and a 2-oxopropyl moiety at position C6 and DiAmHap (N-bridge hapten), which contains acetamido
groups at positions C3 and C6 as well as a δ-aminobutyl group on nitrogen (Figure 8).
Figure 8. PrOxyHap and DiAmHap.
These haptens were conjugated with tetanus toxoid. Mice were immunized and the antibody
titer levels showed the following result: DiAmHap > 6-PrOxyHap. The latter caused the inhibition of
the antinociceptive effects of heroin, morphine and 6-O-acetylmorphine in the animals, facillitated by
the antigens.
Matyas et al. designed further heroin-like haptens [30]: they have synthesized the previously
published HerHap (N-bridge hapten) by Stowe et al.; 6-AcMorHap (C3 hapten) is a 6-O-acetyl-morphine
derivative that contains amino group in position C3 and this group is acylated with
β-mercaptopropionic
acid and MorHap (C6 hapten) is a derivative of 6-β-amino-6-desoxymorphine (Figure 9).
Figure 9. 6-AcMorHap and MorHap.
These haptens were conjugated to tetanus toxoid and they have produced high titers of antibodies
in mice in the following order: MorHap > HerHap >> 6-AcMorHap.

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The DiAmHap contains an isosteric acetamido group instead of an ester. Because of this only one
type of antibody is induced that is reactive with the C3 and C6 acetyl-groups of heroin. In the case of
the ester derivative, a heterogeneous population of antibodies was induced, because of the different
structures of the hydrolyzed metabolites.
It is evident from the afore-mentioned survey, that the structure of the opiate skeleton can
significantly influence the immunization properties of carrier protein coupled haptens. The purpose of
this work was to synthesize N-substituted-4,5-epoxynormorphinans, carrying a free carboxylic group.
We planned to examine the reactions of normorphinans with bromoacetic acid ethyl ester and with
acrylic acid ethyl ester. The hydrolysis yields haptens with free COOH groups which can be connected
with CH2 spacers. These compounds can be considered as morphinan skeleton N-substituted glycines
and morphinan skeleton N-substituted β-amino propionic acids. We designed—for studying—the
coupling of these carboxylic acid containing haptens with amino acid esters in order to model the
coupling reactions of the hapten molecule with carrier proteins which contain free amino groups.
For these novel compounds a detailed NMR analysis was planned.
2. Results and Discussion
2.1. Hapten Synthesis
Hapten molecules were designed with a morphine skeleton which contained free carboxylic groups
on the nitrogen substituent connected with CH2 spacers to the nitrogen. Morphine, dihydromorphine,
codeine, dihydrocodeine, oxycodone and oxymorphone were selected as model compounds and the
syntheses of these hapten type derivatives included N-demethylation and N-alkylation reactions.
For this purpose, the desired normorphine derivatives (normorphine (
), norcodeine ( ), dihydronorcodeine ( ), noroxymorphone ( ) and noroxycodone (
be obtained by the N-demethylation of the starting molecules [31–37].
1
), dihydronormorphine
(2
3
4
5
6) (Figure 10)) can
Figure 10. Normorphine derivatives.
Codeine or dihydrocodeine can be N-demethylated with
α-chloro-ethyl chloroformate in
1,2-dichloroethane solvent and the intermediate carbamate was heated with methanol to yield
the hydrochloride salt of norcodeine (dihydronorcodeine) (Scheme 1).

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Scheme 1. N-demethylation of (dihydro)codeine: (
(b) methanol, heating.
a) α-chloro-ethyl chloroformate, 1,2-dichloroethane;
For the preparation of (dihydro)normorphine 3,6-diacetyl (dihydro)morphine was treated with
-chloro-ethyl chloroformate and after cleavage of the carbamate 3,6-diacetyl (dihydro)normorphine
α
hydrochloride, salt was obtained. Acid hydrolysis of 3,6-diacetyl (dihydro)normorphine yielded
(dihydro)normorphine (Scheme 2).
Scheme 2.
N-demethylation of (dihydro)morphine:
(a) α-chloro-ethyl chloroformate,
1,2-dichloroethane; (b) methanol, heating, acid hydrolysis.
3,14-di-O-Acetyloxymorphone and 14-O-acetyloxycodone were N-demethylated by the above
procedure, and these reactions resulted in 3,14-di-O-acetylnoroxymorphone hydrochloride and
14-O-acetylnoroxycodone hydrochloride. After acid hydrolysis (10% HCl, 6h reflux) noroxymorphone
and noroxycodone were isolated in high yields (Scheme 3).
Scheme 3. N-demethylation of oxymorphone and oxycodone: (a) α-chloro-ethyl chloroformate,
1,2-dichloroethane; (b) methanol, heating, acid hydrolysis.
The next step is the N-alkylation of the norcompounds. Depending on the linker chain size
different methods were selected.
In the case of the methylene bridge, ethyl bromoacetate was used (Scheme 4) and the starting
nor-compound was dissolved in dimethyl formamide (DMF) or acetonitrile in the presence of sodium
hydrogen carbonate, the reaction mixture was heated under reflux for 16 h. The conversion of the reaction
was monitored by thin layer chromatography (TLC). In this reaction the N-carboxymethyl-nor-compound
ethyl esters were obtained.

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Scheme 4. N-alkylation of nor-compounds: ethyl bromoacetate, sodium hydrogen carbonate, acetonitrile
or dimethyl formamide, refl. 16 h.
To synthesize compounds with an ethylene linker, the reaction of ethyl acrylate with
4,5-epoxynormorphinans was studied (Scheme 5). The reaction was performed in ethanol and in the
presence of triethylamine the mixture was heated under reflux for 3 h. The conversion was always
complete and the products N-carboxyethyl-nor-compound ethyl esters were isolated in high yields.
(In case of normorphine: morphinan-17
β-propionic acid-7,8-didehydro-4,5-epoxi-3,6-dihydroxy-ethyl
ester.)
Scheme 5. N-alkylation of nor-compounds: ethyl acrylate, triethylamine, ethanol, refl. 3 h.
Then, the hydrolysis of the aforementioned esters (Scheme 6) with sodium hydroxide in ethanol
at 60 ◦C, was studied. TLC was used to monitor the reaction. After complete conversion, the pH was
adjusted to 3–4 and the mixture was evaporated till dry, to get the hydrochloride salt.

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Scheme 6. Hydrolysis of esters: 1 M NaOH, ethanol, water, heating, 1 h.
In the next step, the reaction of the free carboxylic group containing molecules with glycine ethyl
ester was attempted (Scheme 7). Reagents which are common in peptide synthesis were used like
N,N0-dicyclohexyl carbodiimide (DCCI) or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC)
and 1-hydroxybenzotriazole (HOBt). Unfortunately, these couplings gave very low yields and the
mixtures did not result in the expected compounds. The N-acylated glycine esters were not able to be
isolated. In order to prepare these target compounds, another synthesis route was selected.
Scheme 7. Attempted amino acid coupling: glycine ethyl ester, N,N0-dicyclohexyl carbodiimide (DCCI)
or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), 1-hydroxybenzotriazole (HOBt), water,
room temperature.
In the cases of the methylene bridge-containing compounds, the retro-synthetic analysis revealed
that the linker between the nor-compounds and the glycine ethyl ester can be derivatized with
chloroacetyl chloride and N-alkylation with N-(chloroacetyl)glycine ethyl ester, resulting in the target
compounds (Scheme 8).

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Scheme 8. Amino acid connected hapten: N-chloroacetlyglycine ethyl ester, sodium hydrogen
carbonate, potassium iodide, acetonitrile, 60 ◦C, 8 h.
The nor-compound was treated with the N-(chloroacetyl)glycine ethyl ester in acetonitrile in
the presence of potassium iodide and sodium hydrogen carbonate. The mixture was heated and
stirred at 60 ◦C until conversion was complete. The products were isolated by the usual work-up
and purification of the compounds was performed by column chromatography. These esters were
hydrolyzed using the aforementioned reaction conditions (Scheme 9).
Scheme 9. Hydrolysis of N-acetylglycine esters: 1 M NaOH, ethanol, water, heating, 1 h.
Structures of all the above mentioned compounds were confirmed with 1D and 2D NMR as well as
HR-MS measurements.
2.2. Protonation Constants of the N-Acetylglycine Opioid Compounds
The most important physicochemical properties influencing the pharmacokinetic behavior of
drugs and biomolecules are the acid-base properties, lipophilicity, solubility and permeability, all related
to passive absorption [38].
The acid-base character determines the ionization state of a molecule in a solution of a particular pH.
Consequently, all pharmacokinetic properties, namely absorption, distribution, metabolism, excretion
and toxicity (ADMET) are influenced by the ionization state under varying pH conditions [39].
N-acetylglycine opioid compounds can have up to three basic functional groups, namely an amino
and a phenolate on the main opioid skeleton, and in the glycine side chain a carboxylate group.
Such compounds are tribasic and can be characterized by three protonation constants, K1, K2 and
K3. Compounds with a methoxy group in place of the phenolate are dibasic. Esterification of the
carboxylic site further reduces the number of basic groups, so the esters of codeine derivatives contain
just an amino group as a basic site.
After the synthesis of the new N-acetylglycine opioid compounds, their protonation
macroconstants were also determined to characterize their acid-base properties. The ionization
state of molecules under the diverse conditions of pH in the various parts of the body influences all their

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pharmacokinetic properties during absorption, distribution, metabolism and excretion. The binding
to target molecules (the pharmacodynamic activity), occurs at a definite ionization state. Acid-base
properties play a significant part in the formulation of drug substances for both oral dosage and
intravenous forms as well [40].
We have recently published a review on the site-specific acid-base properties of morphine and
related compounds [41], where it was shown that the basicity of the amino and phenolate site is usually
comparable, with both of them protonating in slightly alkaline solutions. Protonation constants K1 and
K2 of the tribasic compounds describe the uptake of the first two protons on these functional groups.
The basicity of the carboxylate site is smaller by several orders of magnitude, thus K3 values almost
exclusively characterize the protonation of this site.
The main goal of the study of the protonation constants was to see how these newly synthesized
side chains with different electron withdrawing groups (amide, carboxylic acid ester, carboxylate)
influence the basicity of the amino group in the vicinity. A potentially smaller basicity of the amino
group could change the relative concentration of the zwitterionic form compared to the parent
compounds, influencing many pharmacokinetic properties.
pH-potentiometry is the standard method for the determination of protonation constants [38].
This technique was used to obtain the protonation constants of the compounds, with the exception of
the rather small log K3 values where NMR-pH titrations were carried out using indicator molecules to
show the exact pH values in highly acidic media.
Although these compounds contain up to twenty protons connected to carbon atoms, in the acidic
pH region, the chemical shift of only the protons that are located close to the protonating carboxylate
site changes significantly. The signals of the methylene and amide protons in the glycine side chain
were followed, connected to carbon and nitrogen atoms, respectively. None of the other protons
showed a significant change in their chemical shifts in acidic solutions.
The protonation constants are collected in Table 1.
Table 1. Protonation constants of the N-acetylglycine opioid compounds. The standard deviations are
in brackets.
Compounds
logK1
logK2
logK3
N-acetylglycine-normorphine (37)
N-acetylglycine-normorphine ethyl ester (31)
N-acetylglycine-norcodeine (39)
9.27 (0.03)
9.19 (0.05)
5.87 (0.04)
5.53 (0.05)
9.33 (0.04)
6.68 (0.05)
6.21 (0.04)
9.08 (0.05)
8.95 (0.05)
5.83 (0.05)
5.32 (0.03)
6.06 (0.01)
5.53 (0.04)
3.18 (0.01)
-
6.30 (0.03)
3.20 (0.02)
-
5.94 (0.04)
5.44 (0.02)
3.19 (0.02)
-
3.18 (0.02)
-
-
-
-
-
-
N-acetylglycine-norcodeine ethyl ester (33)
N-acetylglycine-dihydronormorphine ethyl ester (32
N-acetylglycine-dihydronorcodeine (40)
N-acetylglycine-dihydronorcodeine ethyl ester (34)
N-acetylglycine-noroxymorphone (41)
N-acetylglycine-noroxymorphone ethyl ester (35)
N-acetylglycine-noroxycodone (42)
N-acetylglycine-noroxycodone ethyl ester (36)
)
3.17 (0.02)
-
-
-
The vicinity of the electron withdrawing amide group significantly decreases the basicity of the
amino group, thus, in these opioid derivatives the basicity of the phenolate site is much larger than that
of the amino site. Consequently, the K1 constant practically characterizes the basicity of the phenolate
site, whereas K2 characterizes that of the amino site, when the phenolate already holds a proton. The K3
constant can be ordered to the carboxylate site when the other two sites are already protonated.
In the morphine-dihydromorphine pairs, hydrogenation of the C7-C8 double bond increases
the electron density and thus, the basicity of both the phenolate and the amino sites. In the
morphine-oxymorphone pairs the replacement of the C6 hydroxyl group by the electron withdrawing
keto group, decreases the basicity of both the phenolate and the amino sites.

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The higher amino basicity of the carboxylic acids compared to their ester derivatives can be
interpreted by the fact that in the pH range of the amino protonation, the carboxyl groups are
predominantly deprotonated, thus negatively charged. Hence they do not have a strong electron
withdrawing effect, unlike the uncharged ester groups. The K3 values, just as the K2 values of the
methoxy derivatives characterize the protonation of the carboxylate sites in acidic medium and are
practically identical in all the investigated compounds. This observation can be explained by the fact
that the carboxylate site lies several covalent bonds away from the opioid skeleton, so any changes
introduced in that skeleton will have practically no effect on the carboxylate basicity.
The determination of protonation constants allows the construction of species distribution
diagrams, shown here for N-acetylglycine normorphine (37), as example (Figure 11). The protonation
state of each group can be seen above the curves.
O-
NH2
COO
OH
NH3
COOH
OH
NH3
COO
OH
NH2
COO
+
+
-
-
-
1.0
0.8
0.6
0.4
0.2
0.0
0
2
4
6
8
10
12
14
pH
Figure 11. The species distribution diagram of N-acetylglycine normorphine.
The intersections of the species distribution curves show that in the pH range 6.06–9.27 this
compound mainly exists in the anionic, in more alkaline solutions in the dianionic form. In slightly
acidic pH values the zwitterionic form has the highest contribution to the mole fraction, whereas in
more acidic solutions the cationic form is dominant.
2.3. Protonation Constants of the N-Carboxyalkyl Opioid Compounds
The side-chain of these molecules does not have an electron withdrawing amide group, thus, the
basicity of the amino and phenolate sites becomes comparable. In such cases protonation microconstants
are needed to describe the basicity of the variously protonated forms [39].
Tribasic N-carboxyalkyl opioid compounds exist in solutions in eight microscopic protonation
forms (Figure 12) and twelve microconstants are needed to describe their protonation microequilibria.
Figure 12. The protonation scheme of tribasic N-carboxyalkyl opioid compounds.

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K1, K2 and K3 are the stepwise macroconstants, small case k stands for the microconstants, indices
C, N and O designate the carbon, nitrogen and oxygen atoms in the carboxylate, amino and phenolate
groups, respectively. Superscripts of microconstants indicate the group protonating in the given
microequilibrium protonation process, whereas the subscript (if any) stands for the group already
holding proton during the process [39].
Some of the relationships between the micro and macroconstants of tribasic N-carboxyalkyl opioid
compounds are as follows:
β1 = K1 = kN + kO + kC
(1)
β3 = K1K2K3 = kOkNkC = kCkCNkCO,N = · · ·
(2)
O
N,O
N-carboxyalkyl opioid compounds with either a methoxy or an ester function are only dibasic,
and exist in solutions in four microscopic protonation forms. Such ligands (abbreviated as L) carry one
negative charge in alkaline solutions and upon the uptake of a proton, they will be transformed into
either a non-charged or zwitterionic form. These two forms are protonation isomers, differing from each
other only in the site of protonation. The uptake of a second proton produces a single cationic species.
These four microspecies, alongside their respective protonation microconstants, are shown in Figure 13,
exemplified by N-carboxyethyl-normorphine ethyl ester (13) and N-carboxyethyl-norcodeine (27).
Figure 13. The microscopic protonation forms and microspecies of N-carboxyethyl-normorphine ethyl
ester (13) and N-carboxyethyl-norcodeine (27).

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2.3.1. Determination of the Protonation Macroconstants
For the determination of protonation macroconstants the same strategy was followed as outlined
above for the N-acetylglycine opioid derivatives. pH-potentiometry was used to obtain the protonation
macroconstants, with the exception of the rather small log K3 values, where NMR-pH titrations were
carried out using indicator molecules. Typically, the doublets of the H9 proton and the signals of
the protons in the ethylene or methylene bridge connecting the amino and carboxylate sites were
followed, unless they were overlapped by other interfering signals. The NMR-pH titration curve of
N-carboxymethyl-noroxymorphone (23) can be seen in Figure 14.
4.10
4.05
H9
H(methylene bridge)
4.00
3.95
3.90
δ
3.85
3.80
3.75
3.70
0.5
1.0
1.5
2.0
2.5
3.0
3.5
pH
Figure 14.
NMR-pH titration curves of the H9 and methylene bridge protons of
N-carboxymethyl-noroxymorphone (23). Computer fits for log K3 are shown in solid lines.
The protonation macroconstants are collected in Table 2.
Table 2. Protonation macroconstants of the N-carboxyalkyl opioid compounds. The standard deviations
are in brackets.
Compounds
logK1
logK2
logK3
N-carboxyethyl-normorphine (25)
N-carboxyethyl-normorphine ethyl ester (13)
N-carboxyethyl-norcodeine (27)
9.54 (0.05)
9.33 (0.08)
8.38 (0.03)
7.34 (0.03)
9.66 (0.06)
9.48 (0.06)
8.76 (0.03)
8.00 (0.04)
9.66 (0.03)
9.15 (0.01)
8.75 (0.06)
7.29 (0.03)
9.38 (0.08)
9.15 (0.08)
8.33 (0.07)
5.64 (0.04)
9.75 (0.08)
9.22 (0.04)
8.86 (0.02)
6.33 (0.02)
9.28 (0.08)
9.05 (0.03)
8.07 (0.06)
5.81 (0.04)
8.17 (0.06)
7.34 (0.01)
3.27 (0.04)
3.27 (0.06)
N-carboxyethyl-norcodeine ethyl ester (15)
N-carboxyethyl-dihydronormorphine (26)
N-carboxyethyl-dihydronormorphine ethyl ester (14)
N-carboxyethyl-dihydronorcodeine (28)
N-carboxyethyl-dihydronorcodeine ethyl ester (16)
N-carboxyethyl-noroxymorphone (29)
N-carboxyethyl-noroxymorphone ethyl ester (17)
N-carboxyethyl-noroxycodone (30)
N-carboxyethyl-noroxycodone ethyl ester (18)
N-carboxymethyl-normorphine (19)
8.51 (0.08)
7.89 (0.03)
3.23 (0.06)
3.23 (0.06)
3.00 (0.07)
1.87 (0.01)
1.90 (0.01)
1.74 (0.01)
8.48 (0.01)
7.29 (0.02)
2.99 (0.04)
8.08 (0.03)
5.56 (0.03)
1.87 (0.01)
N-carboxymethyl-normorphine ethyl ester (7)
N-carboxymethyl-norcodeine (21)
N-carboxymethyl-norcodeine ethyl ester (9)
N-carboxymethyl-dihydronormorphine (20)
N-carboxymethyl-dihydronormorphine ethyl ester (8)
N-carboxymethyl-dihydronorcodeine (22)
N-carboxymethyl-dihydronorcodeine ethyl ester (10)
N-carboxymethyl-noroxymorphone (23)
N-carboxymethyl-noroxymorphone ethyl ester (11)
N-carboxymethyl-noroxycodone (24)
8.53 (0.08)
6.33 (0.01)
1.88 (0.01)
7.98 (0.03)
5.81 (0.08)
1.70 (0.01)
N-carboxymethyl-noroxycodone ethyl ester (12)

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2.3.2. Determination of the Protonation Microconstants of the Dibasic Methoxy Derivatives
Dibasic methoxy derivatives contain an amino and a much less basic carboxylate group, thus the
deductive method was chosen for the determination of microconstants, which requires an appropriate
model compound to mimic the minor species [39]. The macroconstants of the methoxy ester derivatives
were used as the kCN microconstants of the dibasic methoxy derivatives. For dibasic compounds,
in the knowledge of one microconstant and the two macroconstants, all other microconstants can be
calculated (Table 3). Table 3 also contains the logEC,N pair-interactivity parameter that characterizes
how the protonation at one basic site decreases the basicity of the other site.
log EC,N= log kN − log kCN = log kC − log kNC
(3)
Table 3. Protonationmicroconstantsandpair-interactivityparametersoftheinvestigateddibasiccompounds.
Ethers
(27)
(28)
(30)
(21)
(22)
(24)
log kN
log kC
8.38
4.31
7.34
3.27
1.04
8.76
3.99
8.00
3.23
0.76
8.75
4.45
7.29
2.99
1.46
8.33
4.56
5.64
1.87
2.69
8.86
4.41
6.33
1.88
2.53
8.07
3.90
5.81
1.70
2.26
log kCN
log kNC
log EC,N
Esters
(13)
(14)
(17)
(7)
(8)
(11)
log kO
log kN
log kNO
log kON
9.33
8.08
8.59
7.34
9.37
8.54
8.83
8.00
9.15
7.87
8.57
7.29
9.07
6.33
8.38
5.64
9.22
6.95
8.60
6.33
9.05
6.29
8.57
5.81
2.3.3. Determination of the Protonation Microconstants of Tribasic Compounds
In order to determine all the microconstants of the investigated tribasic compounds, the line
of thought detailed below was followed. As already discussed, K3 is practically equal to kNC,O
since protonation along this arrow overwhelmingly predominates the kCO,N and kCN,O processes.
The macroconstants of the methoxy ester derivatives can be inserted in place of the kCN,O microconstants.
With the help of the logEC,N pair-interactivity parameter from Table 3, log kON and log kOC can be obtained.
,
log kCO can be calculated from the macroconstants of the ester derivatives using the value of kCN,O
.
kC and kO can be obtained from β3 using Equation (2). Once these microconstants are known, kN
can be calculated from Equation (1). All the other microconstants can be obtained from Equation
(2) and the logEC,N pair-interactivity parameter. From the microconstants, the logEN,O and logEC,O
pair-interactivity parameters can also be calculated analogous to Equation (3). The results are displayed
in Table 4.

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Table 4. Protonationmicroconstantsandpair-interactivityparametersoftheinvestigateddibasiccompounds.
Compounds
(25)
(26)
(29)
(19)
(20)
(23)
Phenolate Microconstants
log kO
log kNO
log kCO
log kCO,N
9.33
8.59
9.33
8.59
9.41
8.87
9.37
8.83
9.39
8.81
9.15
8.57
9.13
8.44
9.07
8.38
9.42
8.80
9.22
8.60
9.19
8.71
9.05
8.57
Amino Microconstants
log kN
log kON
log kCN
log kCN,O
9.12
8.38
8.08
7.34
9.30
8.76
8.54
8.00
9.33
8.75
7.87
7.29
9.02
8.33
6.33
5.64
9.48
8.86
6.95
6.33
8.55
8.07
6.29
5.81
Carboxylate Microconstants
log kC
4.31
3.27
4.31
3.27
4.03
3.27
3.99
3.23
4.70
3.24
4.46
3.00
4.62
1.93
4.56
1.87
4.63
2.10
4.43
1.90
4.14
1.88
4.00
1.74
log kNC
log kOC
log kNC,O
Pair-Interactivity Parameters
log EC,N
log EN,O
log EC,O
1.04
0.74
0.00
0.76
0.54
0.04
1.46
0.58
0.24
2.69
0.69
0.06
2.53
0.62
0.20
2.26
0.48
0.14
2.3.4. Determination of the Protonation Microconstants of the Dibasic Ester Derivatives
Dibasic ester derivatives contain an amino and phenolate group. As the protonation of the
phenolate group cannot be selectively monitored by UV spectroscopy for morphine derivatives,
the deductive method was chosen for the determination of microconstants. The macroconstant of
the methoxy ester derivative is very close to the kON microconstant of the ester derivative, because
this microconstant lies on the dominant protonation pathway. Thus, the microconstants of the minor
protonation pathway can only be calculated with the help of the logEN,O pair-interactivity parameter
from Table 4. The results are displayed in Table 3.
2.3.5. Discussion of the Protonation Constants of the N-Carboxyalkyl Opioid Compounds
The basicity of the amino and phenolate site is within an order of magnitude in each tribasic
molecule. In the morphine-dihydromorphine pairs, hydrogenation of the C7-C8 double bond increases
the electron density and thus the basicity of both the phenolate and the amino sites.
The higher amino basicity of the carboxylic acids compared to their ester derivatives can once
again be interpreted by the fact that in the pH range of the amino protonation, the negatively charged
carboxylate groups do not have a strong electron withdrawing effect, unlike the uncharged ester groups.
On the other hand, the charge of the side chain of the amino group does not significantly influence the
basicity of the far-away phenolate site, resulting in logEC,O pair-interactivity parameters being close to
zero. kNC,O values that characterize the protonation of the carboxylate sites in acidic medium are much
smaller for N-carboxymethyl than those of the N-carboxyethyl derivatives. This observation can be
explained by the fact that the protonated, electron withdrawing amino group is closer to the carboxylate
in the N-carboxymethyl derivatives, and is further manifested in much higher pair-interactivity
parameters between these two groups.

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The concentration of the various microconstants can be shown in distribution diagrams, shown
here for N-carboxyethyl-normorphine (25) (Figure 15).
1.0
OH
NH3
COOH
O-
NH2
COO
OH
NH3
COO
+
+
0.8
0.6
0.4
0.2
0.0
-
-
OH
NH2
COO
-
-
O-
NH3
COO
+
0
2
4
6
8
10
12
14
pH
Figure 15. The microspecies distribution diagram of N-carboxyethyl-normorphine (25).
The intersections of the species distribution curves show that in the pH range 3.27–8.38 this
compound mainly exists in the zwitterionic form. In more acidic pH values, the cationic form has the
highest contribution to the mole fraction, whereas in more alkaline solutions the anion protonated on
the phenolate site, and finally the dianion is dominant.
3. Materials and Methods
3.1. General Information
The reagents and indicator molecules were purchased from Sigma-Aldrich (St. Louis, MO, USA)
and Alfa Aesar (Haverhill, MA, USA) and used without further purification. Solvents were freshly
1
distilled prior to use and were dried over anhydrous Na2SO4. H and 13C-NMR spectra were recorded
on a 600-MHz Varian VNMRS spectrometer (Varian, Inc., NMR Systems, Palo Alto, CA, USA, Varian
is now part of Agilent Technologies) in DMSO-d6 or D2O solutions;
δ is given in ppm relative to
tetramethylsilane (TMS) as internal standard. H and 13C-NMR signals were assigned on the basis of
one- and two-dimensional homo- and heteronuclear experiments (COSY, TOCSY, HMBC and HSQC).
Melting points were taken on a Stuart SMP-3 apparatus (Global Science NZ Ltd., Auckland, New
Zealand). The high-resolution accurate masses were determined with a Dionex Ultimate 3000 UHPLC
system hyphenated with an Orbitrap Q Exactive Focus Mass Spectrometer equipped with electrospray
ionization (ESI) (Thermo Fischer Scientific, Waltham, MA, USA). Reaction progress was observed
by thin-layer chromatography on commercial silica gel plates (Merck silica gel F254 on aluminum
sheets, Darmstadt, Germany) using different mobile phases. For column chromatography, Kieselgel 60
(particle size 0.040–0.063 mm, ordered from VWR Chemicals, Radnor, PA, USA) was employed.
1
3.2. Hapten Synthesis
Altogether 36 compounds have been synthetized and most of them are new molecules,
only N-carboxymethyl-normorphine ethyl ester, and N-carboxyethyl-norcodeine ethyl ester were
previously synthesized [42
norcodeine, dihydronorcodeine, noroxymorphone and noroxycodone) by N-demethylation with
-chloroethyl chloroformate was accomplished. Nor-compounds were converted to N-carboxyethyl
methyl derivatives with ethyl bromoacetate and nor-compounds were also converted to N-carboxyethyl
,43]. The synthesis of nor-compounds (normorphine, dihydronormorphine,
α

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ethyl derivatives with the reaction of ethyl acrylate. The N-substituted esters were hydrolyzed in
order to obtain compounds with free carboxylic acid on nitrogen. The coupling of these free
carboxylic groups with amino acid esters was attempted but these reactions were unsuccessful and
the expected products were could not be isolated. Therefore, a new reaction was elaborated in order
to obtain hapten coupled amino acid esters. The conversion of all nor-compounds was achieved
by the reaction with N-chloroacetyl amino acid esters. Finally, the alkaline hydrolysis of these
N-acetylglycine-normorphinan ethyl esters was achieved too.
3.2.1. N-Demethylation Reactions
Codeine or dihydrocodeine (3 mmol) was dissolved in dried 1,2-dichloroethane (30 mL),
then sodium hydrogen carbonate (0.84 g) was added to the solution. To this ice-cold mixture,
α
-chloroethyl chloroformate (10 mmol) was added dropwise and the reaction mixture was stirred at
room temperature for 30 min., then heated at 90 ◦C overnight. The resulting suspension was cooled to
room temperature and inorganic salts were filtered off. The filtrate was evaporated under reduced
◦
pressure and the residue was dissolved in methanol and the solution was heated at 60 C for 6 h.
Methanol was removed under reduced pressure to yield a crystalline solid, the hydrochloride salt of
norcodeine or dihydronorcodeine. Free base was liberated with 10% sodium hydroxide (pH = 9) and
was extracted with chloroform. The chloroform extract was dried (sodium sulfate) and evaporated to
result in the nor-compound.
Norcodeine (3 δ 6.60 (d, J = 8.2 Hz,
) yield: 87%, m.p.: 185 ◦C (acetone); 1H-NMR (600 MHz, DMSO-d6)
H-2), 6.44 (d, J = 8.2 Hz, H-1), 5.52 (dp, J = 9.7, 1.5 Hz, H-7), 5.21 (dt, J = 9.8, 2.8 Hz, H-8), 4.64 (dd,
J = 5.9, 1.4 Hz, H-5), 4.09 (dh, J = 5.4, 2.6 Hz, H-6), 3.72 (s, OCH3), 3.46 (dd, J = 5.9, 3.3 Hz, H-9), 2.71
(m, H-10), 2.67 (m, H-16), 2.65 (m, H-10), 2.46 (q, J = 2.8 Hz, H-14), 1.82 (td, J = 12.2, 5.5 Hz, H-15), 1.59
(dt, J = 12.3, 2.4 Hz, H-15); 13C-NMR (150 MHz, DMSO-d6)
δ 147.7 (C4), 141.6 (C3), 133.7 (C7), 131.8
(C12), 129.1 (C8), 128.3 (C11), 118.7 (C1), 113.6 (C2), 93.1 (C5), 66.8 (C6), 56.5 (OCH3), 51.5 (C9), 44.3
(C13), 41.1 (C14), 38.4 (C16), 36.9 (C15), 31.3 (C10)
Dihydronorcodeine (4 δ 6.69 (d,
) yield: 83%, m.p.: 194 ◦C (ethanol); 1H-NMR (600 MHz, DMSO-d6)
J = 8.1 Hz, H-2), 6.53 (d, J = 8.1 Hz, H-1), 4.43 (d, J = 4.9 Hz, H-5), 3.81 (m, H-6), 3.76 (s, OCH3), 3.17 (s,
H-9), 2.81 (dd, J = 18.1, 6.2 Hz, H-10), 2.61 (m, H-10), 2.59 (m, H-16), 2.54 (m, H-16), 2.06 (ddd, J = 11.8,
6.7, 2.8 Hz, H-14), 1.66 (td, J = 12.3, 5.2 Hz, H-15), 1.45 (m, H-15), 1.36 (m, H-8), 1.32 (m, H-7), 1.18 (m,
H-7), 0.83 (tt, J = 11.9, 6.1 Hz, H-8); 13C-NMR (150 MHz, DMSO-d6)
δ 147.5 (C4), 141.2 (C3), 130.9 (C12),
127.8 (C11), 118.5 (C1), 114.4 (C2), 91.1 (C5), 66.3 (C6), 56.7 (OCH3), 52.3 (C9), 43.0 (C13), 39.1 (C14),
38.4 (C16), 38.1 (C15), 30.5 (C10), 26.4 (C7), 20.2 (C8)
(Dihydro)normorphine was prepared by N-demethylation of diacetyl (dihydro)morphine by
means of
α-chloro-ethyl chloroformate using the above-mentioned procedure and the carbamate
was treated with methanol at 60 ◦C to afford the diacetyl (dihydro)normorphine hydrochloride salt.
(Dihydro)normorphine was obtained by hydrolysis of diacetyl (dihydro)normorphine
HCl at 100 C for 4 h. (Dihydro)normorphine base was precipitated with 25% ammonia solution
×
HCl in 5%
◦
(pH = 9–10) and filtered off.
Normorphine (1 δ 6.46 (d, J = 8.0 Hz,
) yield: 84%, m.p.: 273–275 ◦C; 1H-NMR (600 MHz, DMSO-d6)
H-2), 6.35 (d, J = 8.1 Hz, H-1), 5.55 (dp, J = 9.9, 1.5 Hz, H-7), 5.20 (dt, J = 9.8, 2.8 Hz, H-8), 4.67 (dd,
J = 6.1, 1.3 Hz, H-5), 4.07 (dd, J = 6.1, 3.1 Hz, H-6), 3.72 (m, H-9), 2.88 (dd, J = 13.2, 4.7 Hz, H-16), 2.73
(m, H-10), 2.72 (m, H-16), 2.55 (p, J = 2.8 Hz, H-14), 1.92 (td, J = 13.0, 5.0 Hz, H-15), 1.68 (m, H-15), 1.20
(s, H-10); 13C-NMR (150 MHz, DMSO-d6)
δ 146.7 (C4), 139.2 (C3), 134.5 (C7), 130.7 (C12), 127.6 (C8),
124.7 (C11), 119.1 (C1), 117.1 (C2), 92.1 (C5), 66.4 (C6), 51.4 (C9), 43.6 (C13), 39.6 (C14), 37.9 (C16), 35.0
(C15), 29.2 (C10)
Dihydronormorphine (
(d, J = 7.9 Hz, H-2), 6.42 (d, J = 8.0 Hz, H-1), 4.42 (d, J = 4.9 Hz, H-5), 3.81 (ddd, J = 8.6, 4.9, 3.5 Hz,
2 δ 6.53
) yield: 80%, m.p.: 265 ◦C (ethanol); 1H-NMR (600 MHz, DMSO-d6)

Molecules 2020, 25, 4009
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H-6), 3.23 (dd, J = 6.2, 2.8 Hz, H-9), 2.79 (dd, J = 18.2, 6.3 Hz, H-10), 2.64 (m, H-16), 2.62 (m, H-10),
2.57 (m, J-16), 2.07 (ddd, J = 11.6, 6.7, 2.9 Hz, H-14), 1.69 (td, J = 12.5, 5.0 Hz, H-15), 1.48 (ddd, J = 12.5,
3.7, 1.5 Hz, H-15), 1.37 (m, H-8), 1.31 (m, H-7), 1.19 (m, H-7), 0.83 (tt, J = 12.2, 6.2 Hz, H-8); 13C-NMR
(150 MHz, DMSO-d6)
δ 146.4 (C4), 138.4 (C3), 130.3 (C12), 125.4 (C11), 118.4 (C1), 117.2 (C2), 90.5 (C5),
66.4 (C6), 52.3 (C9), 43.1 (C13), 38.6 (C14), 38.3 (C16), 37.7 (C15), 29.9 (C10), 26.3 (C7), 20.1 (C8)
14-O-Acetyloxycodone (2 mmol) was N-demethylated with -chloro-ethyl chloroformate
10 mmol) for 16 h. The reaction was monitored by thin-layer chromatography to check the conversion,
if it was necessary, another 5 mmol portion of -chloro-ethyl chloroformate was added. The carbamate
α
(
α
intermediate was decomposed in methanol to yield the hydrochloride salt of 14-O-acetylnoroxycodone.
Acid hydrolysis in refluxing 10% HCl for 6 h resulted in the hydrochloride salt of noroxycodone.
The free base of noroxycodone was liberated from the acid solution with 10% sodium hydroxide
(pH = 10) and it was extracted with chloroform. The chloroform extract was dried under sodium
sulfate and after evaporation of the solution, afforded the oily noroxycodone, which was rubbed with
diethyl ether to produce crystalline material.
Noroxycodone (6 δ 6.70 (d, J = 8.2 Hz,
) yield: 86%, m.p.: 163–166 ◦C; 1H-NMR (600 MHz, DMSO-d6)
H-2), 6.62 (d, J = 8.2 Hz, H-1), 4.70 (s, H-5), 3.78 (s, OCH3), 2.96 (m, H-10), 2.94 (m, H-9), 2.91 (m, H-7),
2.88 (m, H-10), 2.58 (dd, J = 13.3, 4.7 Hz, H-16), 2.34 (td, J = 12.6, 3.2 Hz, H-16), 2.30 (td, J = 12.0, 4.5 Hz,
H-15), 2.08 (dt, J = 14.1, 3.2 Hz, H-7), 1.74 (ddd, J = 13.6, 5.1, 2.9 Hz, H-8), 1.40 (td, J = 14.1, 3.4 Hz, H-8),
1.14 (dd, J = 12.1, 3.0 Hz, H-15); 13C-NMR (150 MHz, DMSO-d6)
δ 208.9 (C6), 144.9 (C4), 142.3 (C3),
130.3 (C12), 126.8 (C11), 119.5 (C1), 115.1 (C2), 90.5 (C5), 70.1 (C14), 57.3 (C9), 56.8 (OCH3), 51.0 (C13),
37.9 (C16), 36.3 (C7), 32.2 (C10), 32.0 (C8), 30.2 (C15)
N-Demethylation of 3,14-di-O-acetyloxymorphone yielded the hydrochloride salt of
3,14-di-O-acetylnoroxymorphone, which was hydrolyzed in 10% HCl solution. The noroxymorphone
base was precipitated from the acid solution with 25% ammonia solution to yield noroxymorphone
which is pure, for further reactions.
Noroxymorphone (5 δ 6.52 (d, J = 8.1 Hz,
) yield: 81%, m.p.: >280 ◦C; 1H-NMR (600 MHz, DMSO-d6)
H-2), 6.49 (d, J = 8.1 Hz, H-1), 4.64 (s, H-5), 2.97 (m, H-9), 2.91 (m, H-10), 2.90 (m, H-7), 2.83 (m, H-10),
2.62 (dd, J = 13.1, 4.6 Hz, H-16), 2.37 (td, J = 12.8, 3.6 Hz, H-16), 2.29 (dd, J = 12.3, 4.8 Hz, H-15), 2.07
(dt, J = 14.2, 3.2 Hz, H-7), 1.74 (ddd, J = 13.6, 5.2, 2.9 Hz, H-8), 1.42 (td, J = 14.0, 3.5 Hz, H-8), 1.15 (dd,
J = 12.1, 3.2 Hz, H-15); 13C-NMR (150 MHz, DMSO-d6)
δ 209.1 (C6), 143.9 (C4), 139.8 (C3), 130.0 (C12),
124.4 (C11), 119.4 (C1), 117.6 (C2), 89.9 (C5), 70.1 (C14), 57.3 (C9), 50.9 (C13), 37.8 (C16), 36.3 (C7), 31.83
(C10), 31.77 (C8), 30.0 (C15)
3.2.2. General Synthesis of N-Carboxymethyl-Nor-Compound Ethyl Esters
The appropriate normorphine derivative (2 mmol) was dissolved in 30 mL of acetonitrile. In the
presence of sodium bicarbonate (10 mmol) ethyl bromoacetate (2.4 mmol) was added to the solution.
The mixture was stirred and refluxed for 16 h. TLC monitoring showed that conversion was complete.
The inorganic salts were filtered and the solvent was evaporated. Water (30 mL) was added to the
residue and the pH was set around nine with cc. ammonia. Then it was extracted with chloroform
(3
×
25 mL) and after unifying the organic phases, it was dried on sodium sulfate. After filtration
the chloroform was evaporated. If it was necessary, column chromatography was used. (chloroform:
methanol 9:1)
(7
) N-carboxymethyl-normorphine ethyl ester (ethyl 2-((7aR)-7,9-dihydroxy-4,4a,7,7a-tetrahydro-
1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetate) yield: 86%, m.p.: 116 ◦C, HR-MS [M +
H+]: calculated: 358.1649, measured: 358.1636; 1H-NMR (600 MHz, DMSO-d6)
6.40 (d, J = 8.0 Hz,
δ
H-2), 6.31 (d, J = 8.0 Hz, H-1), 5.49 (m, H-7), 5.19 (dt, J = 9.7, 2.8 Hz, H-8), 4.64 (dd, J = 6.1, 1.3 Hz, H-5),
4.09 (dd, J = 6.1, 3.1 Hz, H-6), 4.06 (q, J = 7.1 Hz, ester CH2), 3.39 (s, CH2 bridge), 3.35 (m, H-9), 3.23 (d,
J = 16.5 Hz, CH2 bridge), 2.77 (d, J = 18.5 Hz, H-10) 2.58 (m, H-16), 2.55 (m, H-14), 2.31 (td, J = 12.2, 3.4

Molecules 2020, 25, 4009
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Hz, H-16), 2.26 (dd, J = 18.5, 6.4 Hz, H-10), 1.98 (m, H-15), 1.58 (dt, J = 11.0, 2.2 Hz, H-15), 1.16 (t, J = 7.1
Hz, ester CH3); 13C-NMR (150 MHz, DMSO-d6)
170.9 (ester C=O), 146.6 (C4), 138.9 (C3), 133.8 (C7),
δ
131.3 (C12), 128.6 (C8), 125.7 (C11), 118.9 (C1), 116.7 (C2), 91.9 (C5), 66.7 (C6), 60.4 (ester CH2), 57.2
(C9), 56.5 (CH2 bridge), 44.9 (C16), 43.5 (C13), 40.66 (C14), 35.6 (C15), 22.4 (C10), 14.5 (ester CH3)
(
8
) N-carboxymethyl-dihydronormorphine ethyl ester (ethyl 2-((7aR)-7,9-dihydroxy-4,4a,5,6,7,7a-
hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetate) yield: 84%, m.p.: 86 ◦C,
HR-MS [M + H+]: calculated: 360.1805, measured: 360.1789; 1H-NMR (600 MHz, DMSO-d6)
6.49
δ
(d, J = 8.0 Hz, H-2), 6.38 (d, J = 8.0 Hz, H-1), 4.41 (d, J = 4.8 Hz, H-5), 4.04 (q, J = 7.1 Hz, ester CH2),
3.79 (dd, J = 9.1, 4.4 Hz, H-6), 3.31 (s, (CH2 bridge), 3.15 (d, J = 16.4 Hz, (CH2 bridge), 3.02 (dd, J = 6.0,
2.8 Hz, H-9), 2.71 (d, J = 18.3 Hz, H-10), 2.49 (m, H-16) 2.31 (dd, J = 18.4, 6.0 Hz, H-10), 2.18–2.09 (m,
H-14, H-15), 1.78 (d, J = 5.1 Hz, H-15), 1.44–1.38 (m, H-15), 1.35 (dd, J = 13.4, 6.8 Hz, H-8), 1.28 (ddt,
J = 12.8, 6.5, 3.9 Hz, H-7), 1.15 (t, J = 7.1 Hz, ester CH3), 1.11 (m, H-7), 0.83 (tt, J = 12.5, 6.4 Hz, H-8);
13C-NMR (150 MHz, DMSO-d6)
δ 171.0 (ester C=O), 146.4 (C4), 138.4 (C3), 130.4 (C12), 125.4 (C11),
118.4 (C1), 117.1 (C2), 90.3 (C5), 66.5 (C6), 60.4 (ester CH2), 58.3 (C9), 56.8 (CH2 bridge), 44.9 (C16), 42.2
(C13), 38.2 (C14), 37.4 (C15), 26.0 (C7), 21.9 (C10), 19.9 (C8), 14.6 (ester CH3)
(
9
)
N-carboxymethyl-norcodeine ethyl ester (ethyl 2-((7aR)-7-hydroxy-9-methoxy-4,4a,7,7a-
tetrahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetate) yield: 90%, m.p.: oil, HR-MS
[M + H+]: calculated: 372.1805, measured: 372.1800; 1H-NMR (600 MHz, DMSO-d6)
6.58 (d, J = 8.2 Hz
δ
,
H-2), 6.42 (d, J = 8.2 Hz, H-1), 5.49 (ddt, J = 9.8, 3.2, 1.5 Hz, H-7), 5.19 (dt, J = 9.8, 2.8 Hz, H-8), 4.66 (dd,
J = 6.0, 1.3 Hz, H-5), 4.10 (tq, J = 5.7, 2.7 Hz, H-6), 4.05 (q, J = 7.1 Hz, ester CH2), 3.70 (d, J = 2.2 Hz,
CH2 bridge), 3.69 (s, OCH3), 3.23 (d, J = 16.6 Hz, CH2 bridge), 2.81 (d, J = 18.6 Hz, H-10), 2.57 (dp,
J = 8.8, 3.3, 2.6 Hz, H-16), 2.56 (m, H-14), 2.34–2.25 (m, H-10, H-16), 1.98 (td, J = 12.6, 5.0 Hz, H-15),
1.62–1.53 (m, H-15), 1.16 (t, J = 7.1 Hz, ester CH3); 13C-NMR (150 MHz, DMSO-d6)
δ 170.6 (ester C=O),
147.2 (C4), 141,7 (C3), 133.9 (C7), 131.6 (C12), 128.7 (C8), 127.5 (C11), 118.9 (C1), 113.7 (C2), 92.5 (C5),
67.0 (C6), 60.5 (ester CH2), 57.3 (C9), 56.6 (CH2 bridge), 56.5 (OCH3), 44.9 (C16), 43.5 (C13), 40.6 (C14),
35.6 (C15), 22.6 (C10), 14.6 (ester CH3)
(
10) N-carboxymethyl-dihydronorcodeine ethyl ester (ethyl 2-((7aR)-7-hydroxy-9-methoxy-4,4a,5,6,7,
7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetate) yield: 88%, m.p.: oil,
HR-MS [M + H+]: calculated: 374.1961, measured: 374.1959; 1H-NMR (600 MHz, DMSO-d6)
6.66 (d,
δ
J = 8.2 Hz, H-2), 6.50 (d, J = 8.2 Hz, H-1), 4.43 (dd, J = 7.8, 4.9 Hz, H-5), 4.04 (q, J = 7.1 Hz, ester CH2),
3.79 (tt, J = 8.7, 4.1 Hz, H-6), 3.72 (s, OCH3), 3.32 (s, CH2 bridge), 3.16 (d, J = 16.4 Hz, CH2 bridge),
3.04 (dd, J = 5.9, 2.7 Hz, H-9), 2.75 (d, J = 18.4 Hz, H-10), 2.51–2.48 (m, H-16), 2.34 (dd, J = 18.4, 6.0 Hz
,
H-10), 2.17–2.09 (m, H-16, H-14), 1.79 (td, J = 12.4, 5.0 Hz, H-15), 1.40 (ddd, J = 12.5, 3.6, 1.7 Hz, H-15),
1.34 (dd, J = 13.3, 6.7 Hz, H-8), 1.29 (ddd, J = 13.4, 7.2, 4.2 Hz, H-7), 1.15 (t, J = 7.1 Hz, ester CH3),
1.13–1.08 (m, H-7), 0.84 (tt, J = 11.9, 6.1 Hz, H-8); 13C-NMR (150 MHz, DMSO-d6)
δ 170.9 (ester C=O),
147.4 (C4), 141.3 (C3), 130.7 (C12), 127.3 (C11), 118.4 (C1), 114.4 (C2), 90.7 (C5), 66.3 (C6), 60.3 (ester
CH2), 58.2 (C9), 56.8 (CH2 bridge), 56.7 (OCH3), 44.9 (C16), 42.4 (C13), 38.4 (C14), 37.3 (C15), 26.1 (C7),
22.0 (C10), 19.8 (C8), 14.5 (ester CH3)
(
11) N-carboxymethyl-noroxymorphone ethyl ester (ethyl 2-((7aR)-4a,9-dihydroxy-7-oxo-4,4a,5,6,7,
7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetate) yield: 82%, m.p.: 160 ◦C,
HR-MS [M + H+]: calculated: 374.1598, measured: 374.1588; 1H-NMR (600 MHz, DMSO-d6)
6.53 (d,
δ
J = 8.1 Hz, H-2), 6.50 (d, J = 8.1 Hz, H-1), 4.74 (s, H-5), 4.12–4.06 (m, ester CH2), 3.43 (d, J = 17.1 Hz,
CH2 bridge), 3.31 (d, J = 17.2 Hz, CH2 bridge), 2.97 (d, J = 18.5 Hz, H-10), 2.90 (d, J = 5.9 Hz, H-9),
2.89–2.82 (m, H-7), 2.57 (d, J = 5.9 Hz, H-10), 2.49 (m, H-16), 2.38–2.29 (m, H-15), 2.18 (td, J = 12.0,
3.6 Hz, H-16), 2.06 (dt, J = 14.2, 3.2 Hz, H-7), 1.71 (ddd, J = 13.4, 5.0, 3.0 Hz, H-8), 1.40 (td, J = 14.1,
3.4 Hz, H-8), 1.28–1.22 (m, H-15), 1.18 (t, J = 7.1 Hz, ester CH3); 13C-NMR (150 MHz, DMSO-d6)
(C6), 171.3 (ester C=O), 143.8 (C4), 139.8 (C3), 129.6 (C12), 123.6 (C11), 119.5 (C1), 117.6 (C2), 89.7 (C5),
δ 209.0

Molecules 2020, 25, 4009
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70.4 (C14), 62.8 (C9), 60.7 (ester CH2), 55.6 (CH2 bridge), 50.3 (C13), 43.9 (C16), 36.2 (C7), 31.5 (C8), 30.6
(C15), 24.3 (C10), 14.5 (ester CH3)
(
12) N-carboxymethyl-noroxycodone ethyl ester (ethyl 2-((7aR)-4a-hydroxy-9-methoxy-7-oxo-4,4a,5,6,7,
7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetate) yield: 87%, m.p.: 144 ◦C,
HR-MS [M + H+]: calculated: 388.1755, measured: 388.1752; 1H-NMR (600 MHz, DMSO-d6)
6.72 (d,
δ
J = 8.2 Hz, H-2), 6.63 (d, J = 8.2 Hz, H-1), 4.81 (s, H-5), 4.15–4.03 (m, ester CH2), 3.75 (s, OCH3), 3.44 (d,
J = 17.1 Hz, CH2 bridge), 3.32 (d, CH2 bridge), 3.01 (d, J = 18.6 Hz, H-10), 2.92 (d, J = 5.7 Hz, H-9),
2.87 (td, J = 14.4, 5.0 Hz, H-7), 2.59 (dd, J = 18.7, 5.8 Hz, H-10), 2.52–2.48 (m, H-16), 2.34 (td, J = 12.5
,
5.3 Hz, H-15), 2.17 (td, J = 12.0, 3.6 Hz, H-16), 2.06 (dt, J = 14.1, 3.2 Hz, H-7), 1.72 (ddd, J = 13.5, 5.0,
3.0 Hz, H-8), 1.43–1.34 (m, H-8), 1.30–1.23 (m, H-15), 1.18 (t, J = 7.1 Hz, ester CH3); 13C-NMR (150 MHz,
DMSO-d6) δ 208.8 (C6), 171.3 (ester C=O), 144.8 (C4), 142.4 (C3), 129,9 (C12), 125.8 (C11), 119.7 (C1),
115.2 (C2), 90.2 (C5), 70.3 (C14), 62.8 (C9), 60.7 (ester CH2), 56.8 (OCH3), 55.7 (CH2 bridge), 50.2 (C13),
43.8 (C16), 36.3 (C7), 31.6 (C8), 30.5 (C15), 24.3 (C16), 14.5 (ester CH3)
3.2.3. General Synthesis of N-Carboxymethyl-Nor-Compounds
The N-carboxymethyl-nor-compound ethyl ester (0.5 mmol) was added to the mixture of ethanol
(1 g) and water (2.5 mL). To this solution, sodium hydroxide (1 M, 0.5 mmol) was added and the
reaction mixture was heated for 1 h at 60 ◦C. The hydrolysis reaction was monitored by TLC and upon
complete disappearance of the starting material, the pH was adjusted to 3–4 with 10% HCl solution.
Then the solvent was evaporated to obtain the hydrochloride salt of the desired compound.
(
19) N-carboxymethyl-normorphine HCl (2-((7aR)-7,9-dihydroxy-4,4a,7,7a-tetrahydro-1H-4,12-
methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetic acid hydrochloride) yield: 92%, m.p.: > 270 ◦C
decomp., HR-MS [M + H+]: calculated: 330.1336, measured: 330.1332; 1H-NMR (600 MHz, D2O)
δ
6.61 (d, J = 8.2 Hz, H-2), 6.53 (d, J = 8.2 Hz, H-1), 5.60 (m, H-7), 5.24 (d, J = 10.0 Hz, H-8), 4.92 (d,
J = 6.5 Hz, H-5), 4.27 (m, H-9), 4.25 (m, H-6), 4.00 (m, CH2 bridge) 3.48 (q, J = 7.1 Hz, H-16), 3.11 (d,
J = 20.1 Hz, H-10), 2.99 (d, J = 18.1 Hz, H-14), 2.80 (dd, J = 20.0, 6.8 Hz, H-10), 2.39–2.19 (m, H-15),
2.02 (d, J = 14.6 Hz, H-13); 13C-NMR (150 MHz, D2O)
δ 169.2 (ester C=O), 145.5 (C4), 137.8 (C3), 132.9
(C7), 129.0 (C12), 125.4 (C8), 123.0 (C11), 120.0 (C1), 117.6 (C2), 89.9 (C5), 65.5 (C6), 60.2 (C9), 54.9 (CH2
bridge), 47.1 (C16), 41.8 (C13), 38.1 (C14), 32.2 (C15), 21.9 (C16)
(20) N-carboxymethyl-dihydronormorphine HCl (2-((7aR)-7,9-dihydroxy-4,4a,5,6,7,7a-hexahydro-
1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetic acid hydrochloride) yield: 93%, m.p.:
> 315 ◦C decomp., HR-MS [M + H+]: calculated: 332.1492, measured: 332.1482; 1H-NMR (600 MHz,
D2O)
δ 6.69 (d, J = 7.9 Hz, H-2), 6.62 (d, J = 8.2 Hz, H-1), 4.64 (m, H-5), 4.07–3.83 (m, H-6, H-9, CH2
bridge), 3.34 (dd, J = 13.4, 4.4 Hz, H-16), 3.06 (d, J = 20.2 Hz, H-10), 2.91 (dd, J = 19.9, 6.0 Hz, H-10),
2.82 (ddd, J = 17.3, 13.9, 7.6 Hz, H-16), 2.47 (t, J = 19.4 Hz, H-14), 2.08 (td, J = 13.9, 5.0 Hz, H-15), 1.78
(dd, J = 14.7, 3.7 Hz, H-15), 1.44 (q, J = 6.2 Hz, H-7, H-8), 0.99–0.83 (m, H-8); 13C-NMR (150 MHz, D2O)
δ
168.3 (carboxylic C=O), 145.2 (C4), 137.1 (C3), 128.4 (C12), 122.6 (C11), 119.8 (C1), 117.8 (C2), 89.0
(C5), 66.1 (C6), 61.5 (C9), 55.0 (CH2 bridge), 47.5 (C16), 40.2 (C13), 38.3 (C14), 34.1 (C15), 26.0 (C7), 21.4
(C10), 17.7 (C8)
(
21) N-carboxymethyl-norcodeine HCl (2-((7aR)-7-hydroxy-9-methoxy-4,4a,7,7a-tetrahydro-1H-4,
12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetic acid hydrochloride) yield: 95%, m.p.: > 260 ◦C
decomp., HR-MS [M + H+]: calculated: 344.1492, measured: 344.1481; 1H-NMR (600 MHz, D2O)
δ
6.79 (d, J = 8.3 Hz, H-2), 6.65 (d, J = 8.3 Hz, H1), 5.68–5.54 (m, H-7), 5.33–5.18 (m, H-8), 5.03–4.87 (m,
H-5), 4.39–3.94 (m, H-9, H-6, CH2 bridge), 3.72 (s, OCH3), 3.55–3.40 (m, H-16), 3.16 (d, J = 20.1 Hz,
H-10), 3.10–2.73 (m, H-14, H-10), 2.31 (s, H-15), 2.05 (d, J = 14.9 Hz, H-15); 13C-NMR (150 MHz, D2O)
δ
168.8 (carboxylic C=O), 146.4 (C4), 141.9 (C3), 133.1 (C7), 128.9 (C12), 125.5 (C8), 124.0 (C11), 120.4 (C1),
114.7 (C2), 90.6 (C5), 65.9 (C6), 60.5 (C9), 56.4 (OCH3) 54.8 (CH2 bridge), 46.8 (C16), 41.7 (C13), 38.4
(C14), 32.4 (C15), 21.9

Molecules 2020, 25, 4009
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(22) N-carboxymethyl-dihydronorcodeine HCl (2-((7aR)-7-hydroxy-9-methoxy-4,4a,5,6,7,7a-hexahydro-
1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetic acid hydrochloride) yield: 90%, m.p.:
> 275 ◦C decomp., HR-MS [M + H+]: calculated: 346.1649, measured: 346.1644; 1H-NMR (600 MHz,
D2O) δ 6.85 (d, J = 8.2 Hz, H-2), 6.72 (d, J = 8.2 Hz, H-1), 4.69 (d, J = 5.4 Hz, H-5), 4.16–3.79 (m, H-6,
H-9, CH2 bridge), 3.80–3.70 (s, OCH3), 3.38–3.29 (m, H-16), 3.09 (d, J = 20.2 Hz, H-10), 2.99–2.87 (m,
H-10), 2.80 (t, J = 12.6 Hz, H-16), 2.46 (d, J = 11.6 Hz, H-14), 2.08 (dd, J = 14.7, 10.0 Hz, H-15), 1.78 (d,
J = 14.0 Hz, H-15), 1.45 (d, J = 8.7 Hz, H-7, H-8), 0.98–0.87 (m, H-8); 13C-NMR (150 MHz, D2O)
δ 168.5
(carboxylic C=O), 145.8 (C4), 141.3 (C3), 127.8 (C12), 123.4 (C11), 119.9 (C1), 115.0 (C2), 89.2 (C5), 66.0
(C6), 61.5 (C9), 56.7 (OCH3), 55.1 (CH2 bridge), 47.4 (C16), 40.1 (C13), 38.2 (C14), 34.1 (C15), 26.0 (C7),
21.4 (C10), 17.6 (C8)
(23) N-carboxymethyl-noroxymorphone HCl (2-((7aR)-4a,9-dihydroxy-7-oxo-4,4a,5,6,7,7a-hexahydro-
1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetic acid hydrochloride) yield: 96%, m.p.:
> 290 ◦C decomp., HR-MS [M + H+]: calculated: 346.1285, measured: 346.1283; 1H-NMR (600 MHz,
D2O)
δ 6.70 (d, J = 8.2 Hz, H-2), 6.68 (d, J = 8.3 Hz, H-1), 4.92 (s, H-5), 3.93 (d, J = 16.7 Hz, CH2 bridge),
3.88 (d, J = 6.2 Hz, H-9), 3.69 (d, J = 16.7 Hz, CH2 bridge), 3.29 (d, J = 20.0 Hz, H-10), 3.18 (dd, J = 13.1,
4.9 Hz, H-16), 3.06 (dd, J = 20.1, 6.4 Hz, H-10), 2.89 (td, J = 14.8, 5.1 Hz, H-7), 2.81 (td, J = 13.0, 4.1 Hz,
H-16), 2.65 (td, J = 13.5, 5.0 Hz, H-15), 2.20 (dt, J = 14.9, 3.2 Hz, H-7), 1.96 (ddd, J = 14.6, 5.2, 2.9 Hz, H-8),
1.65 (dd, J = 12.9, 3.7 Hz, H-15), 1.60 (dd, J = 14.6, 3.6 Hz, H-8); 13C-NMR (150 MHz, D2O)
δ 209.3 (C6),
168.8 (carboxylic C=O), 143.0 (C4), 138.5 (C3), 127.2 (C12), 121.7 (C11), 121.1 (C1), 118.7 (C2), 89.2 (C5),
70.7 (C14), 64.9 (C9), 54.7 (CH2 bridge), 48.6 (C13), 46.7 (C16), 34.6 (C7), 30.5 (C8), 27.5 (C15), 23.9 (C10)
(24) N-carboxymethyl-noroxycodone HCl (2-((7aR)-4a-hydroxy-9-methoxy-7-oxo-4,4a,5,6,7,7a-
hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetic acid hydrochloride) yield:
94%, m.p.: > 210 ◦C decomp., HR-MS [M + H+]: calculated: 360.1442, measured: 360.1435; 1H-NMR
(600 MHz, D2O)
δ 6.86 (d, J = 8.4 Hz, H-2), 6.78 (d, J = 8.4 Hz, H-1), 4.95 (s, H-5), 3.94–3.79 (m, H-9,
CH2 bridge), 3.74 (s, OCH3), 3.63 (d, J = 16.4 Hz, CH2 bridge), 3.31 (d, J = 20.0 Hz, H-10), 3.17 (ddt,
J = 13.1, 5.0, 1.4 Hz, H-16), 3.12–3.05 (m, H-10), 2.90 (td, J = 14.8, 5.1 Hz, H-7), 2.80 (td, J = 13.0, 4.1 Hz,
H-16), 2.65 (td, J = 13.5, 5.0 Hz, H-15), 2.21 (dt, J = 14.9, 3.2 Hz, H-7), 1.96 (ddd, J = 14.5, 5.1, 2.9 Hz,
H-8), 1.65 (dd, J = 14.2, 3.8 Hz, H-15), 1.60 (dd, J = 14.6, 3.5 Hz, H-8); 13C-NMR (150 MHz, D2O)
δ 210.3
(C6), 169.2 (carboxylic C=O), 143.9 (C4), 142.4 (C3), 127.1 (C12), 122.5 (C11), 121.1 (C1), 115.6 (C2), 89.4
(C5), 70.4 (C14), 64.9 (C9), 56.7 (OCH3), 55.2 (CH2 bridge), 48.7 (C13), 46.6 (C16), 34.5 (C7), 30.5 (C8),
27.6 (C15), 23.9 (C10)
3.2.4. General Synthesis of N-Carboxyethyl-Nor-Compound Ethyl Esters
The normorphine derivative (2 mmol) was dissolved in 30 mL of ethanol. In the presence of
triethylamine (1 mL) ethyl acrylate (2.4 mmol) was added to the solution. The mixture was stirred and
refluxed for 3 h. After the TLC showed no more starting compound, the solvent was evaporated. If it
was necessary, column chromatography was used. (chloroform:methanol 9:1)
(
13) N-carboxyethyl-normorphine ethyl ester (ethyl 3-((7aR)-7,9-dihydroxy-4,4a,7,7a-tetr◦ahydro-1H-
4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)propanoate) yield: 98%, m.p.: 158 C, HR-MS
[M + H+]: calculated: 372.1805, measured: 372.1798; 1H-NMR (600 MHz, DMSO-d6)
δ 6.44 (d,
J = 8.0 Hz, H-2), 6.34 (d, J = 8.1 Hz, H-1), 5.52 (dp, J = 9.7, 1.5 Hz, H-7), 5.22 (dt, J = 9.8, 2.8 Hz, H-8),
4.66 (dd, J = 6.1, 1.3 Hz, H-5), 4.15–4.00 (m, H-6, ester CH2), 3.35 (m, H-9), 2.78 (dt, J = 14.3, 5.9 Hz, H-10,
ethylene bridge CH2), 2.64 (dt, J = 12.9, 6.6 Hz, ethylene bridge CH2), 2.60–2.53 (m, H-16), 2.48–2.39
(m, H-14, ethylene bridge CH2), 2.33–2.21 (m, H-16, H-10), 1.92 (td, J = 12.5, 5.0 Hz, H-15), 1.61 (ddd,
J = 12.5, 3.4, 1.7 Hz, H-15), 1.19 (t, J = 7.1 Hz, ester CH3); 13C-NMR (150 MHz, DMSO-d6)
δ 172.3 (ester
C=O), 146.8 (C4), 138.7 (C3), 133.9 (C7), 131.2 (C12), 128.8 (C8), 125.7 (C11), 118.9 (C1), 116.8 (C2), 92.0
(C5), 66.8 (C6), 60.3 (ester CH2), 57.1 (C9), 50.8 (ethylene bridge CH2), 44.6 (C16), 43.8 (C13), 41.6 (C14),
36.0 (C15), 33.7 (ethylene bridge CH2), 22.2 (C10), 14.7 (ester CH3)

Molecules 2020, 25, 4009
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(
14) N-carboxyethyl-dihydronormorphine ethyl ester (ethyl 3-((7aR)-7,9-dihydroxy-4,4a,5,6,7,7a-
hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)propanoate) yield: 96%, m.p.:
120 ◦C, HR-MS [M + H+]: calculated: 374.1961, measured: 374.1960; 1H-NMR (600 MHz, DMSO-d6)
δ
6.53 (d, J = 8.0 Hz, H-2), 6.41 (d, J = 8.0 Hz, H-1), 4.44 (d, J = 4.8 Hz, H-5), 4.06 (q, J = 7.1 Hz, ester
CH2), 3.80 (dq, J = 8.7, 4.2 Hz, H-6), 3.16–2.99 (m, H-9), 2.77 (d, J = 18.5 Hz, H-10), 2.46 (d, J = 6.9 Hz,
ethylene bridge CH2), 2.12 (d, J = 39.2 Hz, H-16), 1.82–1.72 (m, H-15), 1.46 (d, J = 12.4 Hz, H-15), 1.38
(dq, J = 13.5, 6.9 Hz, H-8), 1.30 (dtd, J = 13.2, 6.5, 3.4 Hz, H-7), 1.18 (td, J = 7.2, 4.2 Hz, H-7, ester CH3),
0.87 (tt, J = 12.6, 6.4 Hz, H-8); 13C-NMR (150 MHz, DMSO-d6)
δ 172.2 (ester C=O), 146.5 (C4), 138.3
(C3), 130.1 (C12), 125.2 (C11), 117.8 (C1), 116.6 (C2), 89.6 (C5), 65.8 (C6), 59.6 (ester CH2), 57.4 (C9), 49.9
(ethylene bridge CH2), 44.0 (C16), 42.7 (C13), 37.6 (C14), 36.7 (C15), 32.6 (ethylene bridge CH2), 25.4
(C7), 20.9 (C10), 19.3 (C8), 14.2 (ester CH3)
(
15) N-carboxyethyl-norcodeine ethyl ester (ethyl 3-((7aR)-7-hydroxy-9-methoxy-4,4a,7,7a-tetrahydro-
1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)propanoate) yield: 97%, m.p.: oil, HR-MS [M +
H+]: calculated: 386.1962, measured: 386.1951; 1H-NMR (600 MHz, DMSO-d6)
6.61 (d, J = 8.2 Hz,
δ
H-2), 6.45 (d, J = 8.2 Hz, H-1), 5.53 (dp, J = 9.8, 1.5 Hz, H-7), 5.23 (dt, J = 9.8, 2.8 Hz, H-8), 4.67 (dd,
J = 5.9, 1.3 Hz, H-5), 4.11 (dt, J = 5.7, 2.9 Hz, H-6), 4.07 (q, J = 7.1 Hz, ester CH2), 3.71 (s, OCH3), 3.36 (s,
H-9), 2.86–2.74 (m, H-10, ethylene bridge), 2.64 (dt, J = 12.9, 6.7 Hz, ethylene bridge), 2.61–2.53 (m,
H-16), 2.49–2.42 (m, H-14, ethylene bridge), 2.31 (dd, J = 18.6, 6.3 Hz, H-10), 2.24 (td, J = 12.2, 3.4 Hz,
H-16), 1.92 (td, J = 12.5, 5.0 Hz, H-15), 1.61 (ddd, J = 12.6, 3.4, 1.7 Hz, H-15), 1.18 (t, J = 7.1 Hz, ester
CH3); 13C-NMR (150 MHz, DMSO-d6)
δ 171.7 (ester C=O), 145.0 (C4), 141.2 (C3), 133.1 (C7), 130.7
(C12), 128.0 (C8), 127.0 (C11), 118.1 (C1), 113.0 (C2), 91.7 (C5), 66.2 (C6), 59.5 (ester CH2), 56.2 (C9), 55.8
(OCH3), 49.9 (ethylene bridge CH2), 43.7 (C16), 43.4 (C13), 40.2 (C14), 35.2 (C15), 32.9 (ethylene bridge
CH2), 21.5 (C10), 14.0 (ester CH3)
(
16) N-carboxyethyl-dihydronorcodeine ethyl ester (ethyl 3-((7aR)-7-hydroxy-9-methoxy-4,4a,5,6,7,
7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)propanoate) yield: 93%, m.p.: oil,
HR-MS [M + H+]: calculated: 388.2118, measured: 388.2111; 1H-NMR (600 MHz, DMSO-d6)
6.69 (d,
δ
J = 8.2 Hz, H-2), 6.53 (d, J = 8.1 Hz, H-1), 4.45 (dd, J = 6.7, 4.9 Hz, H-5), 4.06 (qd, J = 7.1, 1.2 Hz, ester
CH2), 3.84–3.77 (m, H-6), 3.76 (s, OCH3), 3.04 (dd, J = 5.9, 2.8 Hz, H-9), 2.81–2.70 (m, H-10, ethylene
bridge CH2), 2.59 (dq, J = 12.8, 6.6, 5.9 Hz, ethylene bridge CH2), 2.46–2.38 (m, ethylene bridge CH2),
2.36 (dd, J = 18.4, 6.0 Hz, H-10), 2.12–2.02 (m, H-14, H-16), 1.74 (ddd, J = 14.3, 11.2, 4.9 Hz, H-15), 1.44
(ddd, J = 12.4, 3.6, 1.7 Hz, H-15), 1.37 (dt, J = 13.4, 6.8 Hz, H-8), 1.31 (dq, J = 10.0, 3.5 Hz, H-7), 1.18 (t, J
= 7.1 Hz, ester CH3), 1.16–1.11 (m, H-8), 0.94–0.82 (m, H-7); 13C-NMR (150 MHz, DMSO-d6)
δ 172.4
(ester C=O), 147.4 (C4), 140.9 (C3), 130.7 (C12), 127.2 (C11), 118.4 (C1), 114.4 (C2), 90.7 (C5), 66.3 (C6),
60.1 (ester CH2), 57.9 (C9), 56.7 (OCH3), 50.6 (ethylene bridge CH2), 44.5 (C16), 42.6 (C13), 38.6 (C14),
37.5 (C15), 33.6 (ethylene bridge CH2), 26.1 (C7), 21.6 (C10), 19.8 (C8), 14.6 (ester CH3)
(
17) N-carboxyethyl-noroxymorphone ethyl ester (ethyl 3-((7aR)-4a,9-dihydroxy-7-oxo-4,4a,5,6,7,7a-
hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)propanoate) yield: 99%, m.p.:
137 ◦C, HR-MS [M + H+]: calculated: 388.1754, measured: 388.1740; 1H-NMR (600 MHz, DMSO-d6)
δ
6.56 (d, J = 8.1 Hz, H-2), 6.52 (d, J = 8.1 Hz, H-1), 4.75 (s, H-5), 4.08 (qd, J = 7.1, 2.8 Hz, ester CH2), 2.97
(d, J = 18.5 Hz, H-10), 2.91 (d, J = 5.9 Hz, H-9), 2.90–2.84 (m, H-7), 2.75 (dt, J = 12.7, 7.2 Hz, ethylene
bridge CH2), 2.67 (dt, J = 12.6, 6.2 Hz, ethylene bridge CH2), 2.60–2.53 (m, H-10), 2.53–2.51 (m, H-16),
2.27 (td, J = 12.6, 5.1 Hz, H-15), 2.08 (dt, J = 14.1, 3.2 Hz, H-7), 2.02 (td, J = 12.1, 3.7 Hz, H-16), 1.74
(ddd, J = 13.3, 5.0, 2.9 Hz, H-8), 1.43 (td, J = 14.0, 3.4 Hz, H-8), 1.32–1.25 (m, H-15), 1.21 (t, J = 7.1 Hz,
ester CH3); 13C-NMR (150 MHz, DMSO-d6)
δ 208.7 (C6), 172.0 (ester C=O), 143.4 (C4), 139.4 (C3), 129.4
(C12), 123.3 (C11), 119.0 (C1), 117.2 (C2), 89.3 (C5), 69.8 (C14), 62.6 (C9), 59.9 (ester CH2), 50.1 (C13),
49.7 (ethylene bridge CH2), 42.7 (C16), 35.8 (C7), 32.9 (ethylene bridge CH2), 31.1 (C8), 30.2 (C15), 23.1
(C10), 14.2 (ester CH3)

Molecules 2020, 25, 4009
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(18) N-carboxyethyl-noroxycodone ethyl ester (ethyl 3-((7aR)-4a-hydroxy-9-methoxy-7-oxo-4,4a,5,6,7,
7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)propanoate) yield: 98%, m.p.:
119 ◦C, HR-MS [M + H+]: calculated: 402.1911, measured: 402.1905; 1H-NMR (600 MHz, DMSO-d6)
δ
6.74 (d, J = 8.3 Hz, H-2), 6.66 (d, J = 8.2 Hz, H-1), 4.83 (d, J = 7.8 Hz, H-5), 4.09 (qd, J = 7.1, 2.8 Hz,
ester CH2), 3.78 (s, OCH3), 3.02 (d, J = 18.6 Hz, H-10), 2.94 (d, J = 5.7 Hz, H-9), 2.89 (d, J = 5.0 Hz, H-7),
2.81–2.72 (m, ethylene bridge CH2), 2.68 (dt, J = 12.6, 6.2 Hz, ethylene bridge CH2), 2.59 (dd, J = 18.7,
5.8 Hz, H-10), 2.57–2.52 (m, ethylene bridge CH2), 2.28 (td, J = 12.6, 5.2 Hz, H-15), 2.08 (dt, J = 14.1,
3.2 Hz, H-7), 2.00 (td, J = 12.2, 3.7 Hz, H-16), 1.75 (ddd, J = 13.4, 5.0, 3.0 Hz, H-8), 1.46–1.37 (m, H-8),
1.33–1.26 (m, H-15), 1.21 (t, J = 7.1 Hz, ester CH3); 13C-NMR (150 MHz, DMSO-d6)
δ 208.3 (C6), 172.1
(ester C=O), 144.2 (C4), 141.9 (C3), 129.5 (C12), 125.5 (C11), 118.9 (C1), 114.4 (C2), 89.4 (C5), 69.8 (C14),
62.5 (C9), 59.4 (ester CH2), 55.9 (OCH3), 50.2 (C13), 49.2 (ethylene bridge CH2), 42.7 (C16), 35.5 (C7),
32.4 (ethylene bridge CH2), 30.8 (C8), 29.7 (C15), 22.8 (C10), 13.9 (ester CH3)
3.2.5. General Synthesis of N-Carboxyethyl-Nor-Compounds
The N-carboxyethyl-nor-compound ethyl ester (0.5 mmol) was added to the mixture of ethanol
(1 g) and water (2.5 mL). To this solution, sodium hydroxide (1 M, 0.5 mmol) was added and the
reaction mixture was heated for 1 h at 60 ◦C. The hydrolysis reaction was monitored by TLC and upon
complete disappearance of the starting material the pH was adjusted to 3–4 with 10% HCl solution.
Then, the solvent was evaporated to obtain the hydrochloride salt of the desired compound.
(25
)
N-carboxyethyl-normorphine HCl (3-((7aR)-7,9-dihydroxy-4,4a,7,7a-tetrahydro-1H-4,12-
methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)propanoic acid hydrochloride) yield: 96%, m.p.:
> 290 ◦C decomp., HR-MS [M + H+]: calculated: 344.1492, measured: 344.1483; 1H-NMR (600 MHz,
D2O) δ 6.64 (d, J = 8.2 Hz, H-2), 6.56 (d, J = 8.2 Hz, H-1), 5.62 (d, J = 9.7 Hz, H-7), 5.27 (d, J = 10.1 Hz,
H-8), 4.94 (dd, J = 6.4, 1.3 Hz, H-5), 4.29–4.18 (m, H-6, H-9), 3.48–3.33 (m, H-16, ethylene bridge CH2),
3.13 (d, J = 20.1 Hz, H-10), 2.96 (t, J = 13.2 Hz, H-16), 2.89–2.78 (m, H-14, H-10), 2.71 (td, J = 7.0, 2.7 Hz,
ethylene bridge CH2), 2.22 (t, J = 13.8 Hz, H-15), 2.04 (d, J = 14.3 Hz, H-15); 13C-NMR (150 MHz, D2O)
δ
175.3 (carboxylic C=O), 145.1 (C4), 137.8 (C3), 129.3 (C12), 133.1 (C7), 125.5 (C8), 123.2 (C11), 120.3
(C1), 117.6 (C2), 90.3 (C5), 65.6 (C6), 58.8 (C9), 50.7 (ethylene bridge CH2), 46.0 (C16), 41.8 (C13), 38.1
(C14), 32.5 (C15), 29.6 (ethylene bridge CH2), 21.2 (C10)
(26) N-carboxyethyl-dihydronormorphine HCl (3-((7aR)-7,9-dihydroxy-4,4a,5,6,7,7a-hexahydro-1H-4,
12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)propanoic acid hydrochloride) yield: 96%, m.p.:
> 295 ◦C decomp., HR-MS [M + H+]: calculated: 346.1649, measured: 346.1647; 1H-NMR (600 MHz,
D2O)
δ 6.70 (dd, J = 8.2, 0.9 Hz, H-2), 6.63 (d, J = 8.1 Hz, H-1), 4.65 (d, J = 5.5 Hz, H-5), 4.02 (td, J =
5.9, 2.8 Hz, H-6), 3.91 (dd, J = 6.4, 2.6 Hz, H-9), 3.48–3.32 (m, ethylene bridge CH2), 3.26 (dd, J = 13.1,
5.3 Hz, H-16), 3.08 (d, J = 19.7 Hz, H-10), 2.90 (dd, J = 19.9, 6.1 Hz, H-10), 2.83–2.72 (m, H-16, ethylene
bridge CH2), 2.33 (ddd, J = 11.7, 5.3, 2.8 Hz, H-14), 2.01 (td, J = 13.6, 4.8 Hz, H-15), 1.83–1.76 (m, H-15),
1.51–1.38 (m, H-7, H-8), 1.00–0.86 (m, H-8); 13C-NMR (150 MHz, D2O)
δ 174.2 (carboxylic C=O), 145.1
(C4), 137.3 (C3), 128.1 (C12), 122.4 (C11), 119.8 (C1), 117.9 (C2), 88.9 (C5), 66.0 (C6), 60.0 (C9), 50.0
(ethylene bridge CH2), 46.9 (C16), 40.6 (C13), 38.3 (C14), 34.0 (C15), 29.0 (ethylene bridge CH2), 25.9
(C7), 20.5 (C10), 17.6 (C8)
(27) N-carboxyethyl-norcodeine HCl (3-((7aR)-7-hydroxy-9-methoxy-4,4a,7,7a-tetrahydro-1H-4,12-
methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)propanoic acid hydrochloride) yield: 93%, m.p.:
> 200 ◦C decomp., HR-MS [M + H+]: calculated: 358.1649, measured: 358.1634; 1H-NMR (600
MHz, D2O)
δ 6.78 (d, J = 8.3 Hz, H-2), 6.65 (d, J = 8.3 Hz, H-1), 5.61 (d, J = 9.8 Hz, H-7), 5.26 (d,
J = 9.9 Hz, H-8), 4.94 (dd, J = 6.4, 1.3 Hz, H-5), 4.32–4.15 (m, H-6, H-9), 3.71 (s, OCH3), 3.51 (q, J = 7.1
Hz, ethylene bridge CH2), 3.48–3.34 (m, H-16), 3.17 (d, J = 20.3 Hz, H-10), 3.00–2.78 (m, H-16, H-14,
H-10), 2.75 (td, J = 7.0, 2.5 Hz, ethylene bridge CH2), 2.22 (d, J = 13.9 Hz, H-15), 2.03 (d, J = 14.4 Hz,
H-15); 13C-NMR (150 MHz, D2O)
δ
175.1 (carboxylic C=O), 146.3 (C4), 142.0 (C3), 133.1 (C7), 129.00
(C12), 125.6 (C8), 124.0 (C11), 120.3 (C1), 114.4 (C2), 90.5 (C5), 65.7 (C6), 58.9 (C9), 56.4 (OCH3), 50.6

Molecules 2020, 25, 4009
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(ethylene bridge CH2), 46.1 (C16), 41.9 (C13), 38.3 (C14), 32.5 (C15), 29.4 (ethylene bridge CH2), 21.2
(C10)
(28) N-carboxyethyl-dihydronorcodeine HCl (3-((7aR)-7-hydroxy-9-methoxy-4,4a,5,6,7,7a-hexahydro-
1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)propanoic acid hydrochloride) yield: 95%, m.p.:
> 220 ◦C decomp., HR-MS [M + H+]: calculated: 360.1805, measured: 360.1791; 1H-NMR (600 MHz,
D2O)
δ 6.82 (d, J = 8.2 Hz, H-2), 6.70 (d, J = 8.2 Hz, H-1), 4.64 (m, H-5), 3.99 (q, J = 4.9 Hz, H-6), 3.92 (dd,
J = 6.3, 2.6 Hz, H-9), 3.73 (s, OCH3), 3.43 (dt, J = 14.1, 7.2 Hz, ethylene bridge CH2), 3.37 (dt, J = 13.7, 7.0
Hz, ethylene bridge CH2), 3.25 (dd, J = 13.2, 4.7 Hz, H-16), 3.10 (d, J = 19.8 Hz, H-10), 2.90 (dd, J = 19.9
6.2 Hz, H-10), 2.79 (td, J = 7.1, 4.4 Hz, ethylene bridge CH2), 2.71 (td, J = 13.3, 4.1 Hz, H-16), 2.35 (ddd,
J = 12.0, 5.4, 2.7 Hz, H-14), 2.03 (td, J = 13.7, 4.8 Hz, H-15), 1.77–1.68 (m, H-15), 1.48–1.34 (m, H-8, H-7),
,
0.90 (tt, J = 11.3, 6.0 Hz, H-8); 13C-NMR (151 MHz, D2O)
δ 173.8 (carboxylic C=O), 146.0 (C4), 141.5
(C3), 128.1 (C12), 123.5 (C11), 119.9 (C1), 114.9 (C2), 89.2 (C5), 66.0 (C6), 60.1 (C9), 56.6 (OCH3), 49.9
(ethylene bridge CH2), 46.9 (C16), 40.5 (C13), 38.1 (C14), 34.1 (C15), 28.9 (ethylene bridge CH2), 25.8
(C7), 20.6 (C10), 17.6 (C8)
(29) N-carboxyethyl-noroxymorphone HCl (3-((7aR)-4a,9-dihydroxy-7-oxo-4,4a,5,6,7,7a-hexahydro-
1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)propanoic acid hydrochloride) yield: 99%, m.p.:
> 280 ◦C decomp., HR-MS [M + H+]: calculated: 360.1442, measured: 360.1332; 1H-NMR (600 MHz,
D2O)
δ 6.72 (d, J = 8.2 Hz, H-2), 6.69 (d, J = 8.3 Hz, H-1), 4.93 (s, H-5), 3.74 (d, J = 6.1 Hz, H-9), 3.41
(hept, J = 6.8, 5.9 Hz, ethylene bridge CH2), 3.34–3.25 (m, H-16, H-10), 3.05 (dd, J = 19.9, 6.3 Hz, H-10),
2.89 (td, J = 14.8, 5.1 Hz, H-7), 2.85–2.66 (m, H-16, ethylene bridge CH2), 2.60 (td, J = 13.4, 4.8 Hz, H-15),
2.21 (dt, J = 14.8, 3.2 Hz, H-7), 1.95 (ddd, J = 14.5, 5.2, 3.0 Hz, H-8), 1.64 (ddd, J = 17.9, 13.7, 3.5 Hz,
H-8, H-15); 13C-NMR (151 MHz, D2O)
δ 211.5 (C6), 175.0 (carboxylic C=O) 143.2 (C4), 138.7 (C3), 127.2
(C12), 121.7 (C11), 120.9 (C1), 118.7 (C2), 89.5 (C5), 70.6 (C14), 64.0 (C9), 49.7 (ethylene bridge CH2),
49.1 (C13), 45.8 (C16), 34.4 (C7), 30.5 (C8), 28.2 (ethylene bridge CH2), 27.1 (C15), 23.1 (C10)
(30) N-carboxyethyl-noroxycodone HCl (3-((7aR)-4a-hydroxy-9-methoxy-7-oxo-4,4a,5,6,7,7a-hexahydro-
1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)propanoic acid hydrochloride) yield: 96%, m.p.:
> 260 ◦C decomp., HR-MS [M + H+]: calculated: 374.1598, measured: 374.1597; 1H-NMR (600 MHz,
D2O) δ 6.87 (d, J = 8.3 Hz, H-2), 6.78 (d, J = 8.4 Hz, H-1), 4.95 (s, H-5), 3.75 (d, J = 4.5 Hz, H-9, OCH3),
3.42 (t, J = 6.6 Hz, ethylene bridge CH2), 3.32 (d, J = 20.1 Hz, H-10, H-16), 3.08 (dd, J = 20.0, 6.3 Hz,
H-10), 2.89 (td, J = 14.8, 5.1 Hz, H-7), 2.82 (dt, J = 18.2, 6.6 Hz, ethylene bridge CH2), 2.70 (td, J = 13.5,
12.9, 4.5 Hz, H-16), 2.60 (td, J = 13.4, 4.8 Hz, H-15), 2.20 (dt, J = 14.9, 3.2 Hz, H-7), 1.95 (ddd, J = 14.6,
5.2, 3.0 Hz, H-8), 1.68–1.59 (m, H-8, H-15); 13C-NMR (151 MHz, D2O)
δ 211.0 (C6), 174.9 (carboxylic
C=O), 144.3 (C4), 142.6 (C3), 127.1 (C12), 122.4 (C11), 121.1 (C1), 115.6 (C2), 89.4 (C5), 70.2 (C14), 64.0
(C9), 56.7 (OCH3), 49.7 (ethylene bridge CH2), 48.4 (C13), 45.9 (C16), 34.4 (C7), 30.5 (C8), 28.2 (ethylene
bridge CH2), 27.2 (C15), 23.1 (C10)
3.2.6. General Synthesis of N-Acetylglycine-Nor-Compound Ethyl Esters
The normorphine derivative (2 mmol) was dissolved in 30 mL of acetonitrile. In the presence of
sodium bicarbonate (10 mmol) and potassium iodide (2.4 mmol), N-(chloroacetyl)glycine ethyl ester
(2.4 mmol) was added to the solution. The mixture was stirred and refluxed for 8 h. After the TLC
showed no more starting compound, the inorganic salts were filtered and the solvent was evaporated.
If it was necessary, column chromatography was used. (chloroform: methanol 9:1)
(
31) N-acetylglycine-normorphine ethyl ester (ethyl 2-(2-((7aR)-7,9-dihydroxy-4,4a,7,7a-tetrahydro-1H-
4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetamido)acetate) yield: 94%, m.p.: 105 ◦C, HR-MS
[M + H+]: calculated: 415.1863, measured: 415.1848; 1H-NMR (600 MHz, DMSO-d6)
H-2), 6.32 (d, J = 8.1 Hz, H-1), 5.53 (dp, J = 9.9, 1.6 Hz, H-7), 5.20 (dt, J = 9.8, 2.8 Hz, H-8), 4.67 (dd, J = 6.1
δ
6.42 (d, J = 8.0 Hz
,
,
1.3 Hz, H-5), 4.06 (q, J = 7.1 Hz, ester CH2), 3.85 (qd, J = 17.4, 6.2 Hz, glycine CH2), 3.35 (dd, J = 6.1, 3.0
Hz, H-9), 3.20 (d, J = 16.1 Hz, CH2 bridge), 2.98 (d, J = 16.1 Hz, CH2 bridge), 2.76 (d, J = 18.4 Hz, H-10),

Molecules 2020, 25, 4009
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2.71 (dd, J = 4.7, 2.5 Hz, H-14), 2.55 (dd, J = 12.1, 4.6 Hz, H-16), 2.41–2.29 (m, H-10, H-16), 2.05 (td,
J = 12.5, 4.9 Hz, H-15), 1.61 (d, J = 12.7 Hz, H-15), 1.16 (t, J = 7.1 Hz, ester CH3); 13C-NMR (150 MHz,
DMSO-d6)
δ 171.0 (amide C=O), 170.3 (ester C=O), 146.7 (C4), 139.1 (C3), 133.8 (C7), 131.2 (C12), 128.7
(C8), 125.4 (C11), 119.0 (C1), 116.7 (C2), 91.8 (C6), 66.9 (C6), 60.8 (ester CH2), 58.9 (CH2 bridge), 57.6
(C9), 45.0 (C16), 43.5 (C13), 40.9 (glycine CH2), 40.7 (C14), 35.7 (C15), 23.3 (C10), 14.5 (ester CH3)
(
32) N-acetylglycine-dihydronormorphine ethyl ester (ethyl 2-(2-((7aR)-7,9-dihydroxy-4,4a,5,6,7,7a-
hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetamido)acetate) yield: 91%, m.p.:
76 ◦C, HR-MS [M + H+]: calculated: 417.2020, measured: 417.2020; 1H-NMR (600 MHz, DMSO-d6)
δ
6.53 (d, J = 8.0 Hz, H-2), 6.42 (d, J = 8.0 Hz, H-1), 4.47 (d, J = 4.8 Hz, H-5), 4.09 (q, J = 7.1 Hz, ester CH2),
3.88 (d, J = 6.4 Hz, glycine CH2), 3.85 (d, J = 6.0 Hz, glycine CH2), 3.83–3.78 (m, H-6), 3.19 (d, J = 16.1
Hz, CH2 bridge), 3.07 (d, J = 3.2 Hz, H-9), 2.93 (d, J = 16.0 Hz, CH2 bridge), 2.73 (d, J = 18.3 Hz, H-10),
2.48 (m, H-16), 2.38 (t, J = 1.8 Hz, H-10), 2.29 (s, H-14), 2.26–2.18 (m, H-16), 1.90 (td, J = 12.3, 4.9 Hz,
H-15), 1.46 (dt, J = 10.8, 2.3 Hz, H-15), 1.40 (dq, J = 13.8, 7.1 Hz, H-8), 1.32 (td, J = 6.7, 3.6 Hz, H-7), 1.18
(m, ester CH3, H-7), 0.86 (d, J = 18.8 Hz, H-8); 13C-NMR (150 MHz, DMSO-d6)
δ 171.0 (amide C=O),
170.6 (ester C=O), 146.5 (C4), 138.5 (C3), 130.4 (C12), 125.3 (C11), 118.5 (C1), 117.2 (C2), 90.4 (C5), 66.7
(C6), 61.0 (ester CH2), 59.2 (CH2 bridge), 58.8 (C9), 45.1 (C16), 42.5 (C13), 41.0 (glycine CH2), 38.2 (C14),
37.5 (C15), 26.0 (C7), 23.0 (C10), 20.0 (C8), 14.6 (ester CH3)
(
33) N-acetylglycine-norcodeine ethyl ester (ethyl 2-(2-((7aR)-7-hydroxy-9-methoxy-4,4a,7,7a-
tetrahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetamido)acetate) yield: 90%, m.p.:
oil, HR-MS [M + H+]: calculated: 429.2020, measured: 429.2011; 1H-NMR (600 MHz, DMSO-d6)
6.59
δ
(d, J = 8.2, H-2), 6.44 (d, J = 8.3, H-1), 5.53 (d, J = 9.8 Hz, H-7), 5.21 (dt, J = 7.4, 3.4 Hz, H-8), 4.69 (d,
J = 5.4 Hz, H-5), 4.10 (m, H-6), 4.06 (tdd, J = 7.2, 4.7, 2.6 Hz, ester CH2), 3.86 (m, glycine CH2), 3.69
(d, J = 3.2 Hz, OCH3), 3.37 (dt, J = 7.6, 3.8 Hz, H-9), 3.20 (dd, J = 16.3, 4.2 Hz, CH2 bridge), 2.98 (dd,
J = 15.7, 3.8 Hz, CH2 bridge), 2.80 (dd, J = 18.5, 4.1 Hz, H-10), 2.75–2.69 (m, H-14), 2.58 (m, H-16),
2.41–2.31 (m, H-10, H-16), 2.11–1.99 (m, H-15), 1.61 (d, J = 12.9 Hz, H-15), 1.16 (ddd, J = 7.1, 4.7, 2.3 Hz,
ester CH3); 13C-NMR (150 MHz, DMSO-d6)
δ 170.9 (amide C=O), 170.5 (ester C=O), 147.7 (C4), 141.8
(C3), 133.7 (C7), 131.5 (C12), 128.7 (C8), 127.5 (C11), 118.8 (C1), 113.8 (C2), 92.4 (C5), 67.0 (C6), 60.9
(ester CH2), 58.9 (CH2 bridge), 55.5 (OCH3), 57.7 (C9), 44.9 (C16), 43.6 (C13), 40.9 (glycine CH2), 40.6
(C14), 35.7 (C15), 23.4 (C10), 14.5 (ester CH3)
(34) N-acetylglycine-dihydronorcodeine ethyl ester (ethyl 2-(2-((7aR)-7-hydroxy-9-methoxy-4,4a,5,6,7,
7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetamido)acetate) yield: 89%
◦
1
m.p.: 76 C, HR-MS [M + H+]: calculated: 431.2177, measured: 431.2165; H-NMR (600 MHz,
DMSO-d6) 6.70 (d, J = 8.2 Hz, H-2), 6.54 (d, J = 8.2 Hz, H-1), 4.49 (dd, J = 4.9, 3.6 Hz, H-5), 4.08
δ
(q, J = 7.1 Hz, ester CH2), 3.88 (d, J = 6.3 Hz, glycine CH2), 3.84 (d, J = 5.9 Hz, glycine CH2), 3.82 (q,
J = 3.9 Hz, H-6), 3.76 (s, OCH3), 3.20 (d, J = 16.1 Hz, CH2 bridge), 3.09 (dd, J = 6.1, 2.7 Hz, H-9), 2.93 (d,
J = 16.1 Hz, CH2 bridge), 2.77 (d, J = 18.4 Hz, H-10), 2.48–2.40 (m, H-10), 2.30 (ddd, J = 11.5, 6.7, 2.7 Hz,
H-14), 2.20 (td, J = 12.2, 3.4 Hz, H-16), 1.90 (td, J = 12.4, 4.9 Hz, H-15), 1.45 (dd, J = 11.6, 3.1 Hz, H-15),
1.39 (dt, J = 13.4, 6.8 Hz, H-8), 1.32 (qd, J = 6.8, 4.7 Hz, H-7), 1.18 (t, J = 7.1 Hz, ester CH3), 0.89 (tt,
J = 12.7, 6.4 Hz, H-8); 13C-NMR (150 MHz, DMSO-d6)
δ 171.0 (amide C=O), 170.4 (ester C=O), 147.4
(C4), 141.3 (C3), 130.6 (C12), 127.2 (C11), 118.5 (C1), 114.5 (C2), 90.7 (C5), 66.4 (C6), 60.8 (ester CH2),
59.1 (CH2 bridge), 58.6 (C9), 55.9 (OCH3), 45.0 (C16), 42.3 (C13), 40.9 (glycine CH2), 38.3 (C14), 37.3
(C15), 26.1 (C7), 22.9 (C10), 19.8 (C8), 14.5 (ester CH3)
(
35) N-acetylglycine-noroxymorphone ethyl ester (ethyl 2-(2-((7aR)-4a,9-dihydroxy-7-oxo-4,4a,5,6,7,7a-
hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetamido)acetate) yield: 95%, m.p.:
83 ◦C, HR-MS [M + H+]: calculated: 431.1813, measured: 431.1799; 1H-NMR (600 MHz, DMSO-d6)
δ
6.56 (d, J = 8.1 Hz, H-2), 6.53 (d, J = 8.1 Hz, H-1), 4.77 (s, H-5), 4.09 (qd, J = 7.1, 1.2 Hz, ester CH2),
3.95 (d, J = 6.7 Hz, glycine CH2), 3.80 (d, J = 5.7 Hz, glycine CH2), 3.23 (d, J = 16.0 Hz, CH2 bridge),
3.02–2.89 (m, CH2 bridge, H-9, H-7), 2.59–2.53 (m, H-16), 2.53–2.51 (m, H-10), 2.43 (td, J = 12.6, 5.0 Hz,

Molecules 2020, 25, 4009
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H-15), 2.08 (dt, J = 14.3, 3.2 Hz, H-7), 1.99 (td, J = 12.0, 3.7 Hz, H-16), 1.73 (ddd, J = 13.3, 5.2, 3.0 Hz,
H-8), 1.29 (dt, J = 11.9, 2.7 Hz, H-15), 1.18 (t, J = 7.1 Hz, ester CH3); 13C-NMR (150 MHz, DMSO-d6)
δ
209.3 (C6), 170.5 (amide C=O), 170.4 (ester C=O), 143.8 (C4), 139.8 (C3), 130.1 (C12), 123.9 (C11), 119.5
(C1), 117.6 (C2), 89.7 (C5), 70.7 (C14), 64.0 (C9), 60.9 (ester CH2), 59.5 (CH2 bridge), 50.1 (C13), 44.2
(C16), 40.9 (glycine CH2), 36.2 (C7), 31.6 (C8), 29.8 (C15), 24.5 (C10), 14.5 (ester CH3)
(36) N-acetylglycine-noroxycodone ethyl ester (ethyl 2-(2-((7aR)-4a-hydroxy-9-methoxy-7-oxo-4,4a,5,6,7,
7a-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetamido)acetate) yield: 96%
◦
1
m.p.: 81 C, HR-MS [M + H+]: calculated: 445.1969, measured: 445.1964; H-NMR (600 MHz,
DMSO-d6) 6.75 (d, J = 8.2 Hz, H-2), 6.67 (d, J = 8.2 Hz, H-1), 4.85 (s, H-5), 4.09 (qd, J = 7.1, 1.4 Hz, ester
δ
CH2), 3.94 (dd, J = 17.3, 6.7 Hz, glycine CH2), 3.79 (s, glycine CH2), 3.78 (s, OCH3), 3.24 (d, J = 16.0 Hz,
CH2 bridge), 3.08–2.92 (m, CH2 bridge, H-7, H-10), 2.65–2.54 (m, H-10, H-16), 2.40–2.37 (m, H-15), 2.08
(dt, J = 14.1, 3.1 Hz, H-7), 1.97 (td, J = 12.0, 3.7 Hz, H-16), 1.75 (ddd, J = 13.3, 5.1, 2.9 Hz, H-8), 1.51–1.37
(m, H-8), 1.32–1.27 (m, H-15), 1.19 (t, J = 7.1 Hz, ester CH3); 13C-NMR (150 MHz, DMSO-d6)
δ 209.0
170.4 (amide C=O), 170.3 (ester C=O), (C6), 142.5 (C3), 144.8 (C4), 130.3 (C12), 126.1 (C11), 119.8 (C1),
115.3 (C2), 90.1 (C5), 70.6 (C14), 60.9 (ester CH2), 59.4 (CH2 bridge), 56.8 (OCH3), 50.2 (C13), 44.1 (C16),
40.7 (glycine CH2), 36.0 (C7), 31.8 (C8), 63.9 (C9), 29.4 (C15), 24.6 (C10), 14.5 (ester CH3)
3.2.7. General Synthesis of N-Acetylglycine-Nor-Compounds
The N-acetylglycine-nor-compound ethyl ester (0.5 mmol) was dissolved in the mixture of ethanol
(1 g) and water (2,5 mL). The mixture was hydrolyzed with sodium hydroxide (1 M, 0.5 mmol) for
1 h at 60 ◦C. The hydrolysis reaction was monitored by TLC and upon complete disapparance of the
starting material, the pH was adjusted to 3–4 with 10% HCl solution. Then the solvent was evaporated
to obtain the hydrochloride salt of the desired compound.
(37) N-acetylglycine-normorphine HCl (2-(2-((7aR)-7,9-dihydroxy-4,4a,7,7a-tetrahydro-1H-4,12-
methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetamido)acetic acid hydrochloride) yield: 98%, m.p.: >
205 ◦C decomp., HR-MS [M + H+]: calculated: 387.1551, measured: 387.1547; 1H-NMR (600 MHz, D2O)
δ
6.65 (d, J = 8.1 Hz, H-2), 6.57 (d, J = 8.2 Hz, H-1), 5.63 (dd, J = 9.8, 2.8 Hz, H-7), 5.25 (d, J = 10.0 Hz,
H-8), 4.96 (dd, J = 6.4, 1.2 Hz, H-5), 4.33–4.00 (m, H-6, H-9), 3.94 (s, glycine CH2), 3.51 (d, J = 7.1 Hz,
CH2 bridge), 3.38 (s, H-16), 3.15 (d, J = 20.1 Hz, H-10), 3.02 (s, H-14), 2.42–2.23 (m, H-15), 2.05 (d,
J = 14.4 Hz, H-15); 13C-NMR (150 MHz, D2O)
δ 173.1 (carboxylic C=O), 165.3 (amide C=O), 145.5 (C4),
138.0 (C3), 133.1 (C7), 129.2 (C12), 125.5 (C8), 123.0 (C11), 120.3 (C1), 117.7 (C2), 90.3 (C5), 65.2 (C6),
60.4 (C9), 57.6 (CH2 bridge), 47.1 (C16), 41.7 (C13), 41.2 (glycine CH2), 38.0 (C14), 32.2 (C15), 22.0 (C10)
(38) N-acetylglycine-dihydronormorphine HCl (2-(2-((7aR)-7,9-dihydroxy-4,4a,5,6,7,7a-hexahydro-1H-
4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetamido)acetic acid hydrochloride) yield: 94%,
m.p.: > 225 ◦C decomp., HR-MS [M + H+]: calculated: 389.1707, measured: 389.1765; 1H-NMR (600
MHz, D2O) δ 6.83 (d, J = 8.3 Hz, H-2), 6.65 (d, J = 8.3 Hz, H-1), 4.74 (d, J = 5.6 Hz, H-5), 4.41–4.17 (m,
H-6, H-9), 4.16–4.00 (m, H-6, H-9), 3.96 (d, J = 3.6 Hz, glycine CH2) 3.31 (d, J = 14.0 Hz, H-16), 3.11 (t,
J = 19.5 Hz, H-10), 2.89 (d, J = 13.5 Hz, CH2 bridge), 2.53 (m, H-14), 2.11 (s, H-15), 1.81 (d, J = 13.0 Hz,
H-15), 1.47 (m, H-8), 0.95 (m, H-8); 13C-NMR (150 MHz, D2O) δ 172.9 (carboxylic C=O), 165.3 (amide
C=O), 145.6 (C4), 138.5 (C3), 128.7 (C12), 123.2 (C11), 120.0 (C1), 117.1 (C2), 89.7 (C5), 66.1 (C6), 61.1
(C9), 57.0 (CH2 bridge), 47.8 (C16), 41.4 (glycine CH2), 41.2 (C13), 38.2 (C14), 32.9 (C15), 25.7 (C7), 21.6
(C10), 17.9 (C8)
(39) N-acetylglycine-norcodeine HCl (2-(2-((7aR)-7-hydroxy-9-methoxy-4,4a,7,7a-tetrahydro-1H-4,12-
methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetamido)acetic acid hydrochloride) yield: 99%, m.p.:
> 200 ◦C decomp., HR-MS [M + H+]: calculated: 401.1707, measured: 401.1704; 1H-NMR (600 MHz,
D2O) δ 6.80 (d, J = 8.3 Hz, H-2), 6.66 (d, J = 8.3 Hz, H-1), 5.62 (d, J = 9.9 Hz, H-7), 5.25 (d, J = 10.0 Hz,
H-8), 4.97 (d, J = 6.3 Hz, H-5), 4.29–4.23 (m, H-6, H-9), 4.16–4.06 (m, CH2 bridge, glycine CH2), 3.72 (s,
OCH3), 3.39 (m, H-16), 3.20–3.17 (m, H-10, H-16), 3.03 (m, H-14), 2.33 (t, J = 13.0 Hz, H-15), 2.04 (d,

Molecules 2020, 25, 4009
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J = 14.5 Hz, H-15); 13C-NMR (150 MHz, D2O)
δ
173.2 (carboxylic C=O), 165.4 (amide C=O), 146.3 (C4),
142.2 (C3), 133.0 (C7), 128.8 (C12), 125.2 (C8), 123.8 (C11), 120.2 (C1), 114.7 (C2), 90.7 (C5), 65.7 (C6),
60.4 (C9), 56.8 (OCH3), 54.5 (CH2 bridge), 47.1 (C16), 41.3 (glycine CH2), 41.9 (C13), 37.9 (C14), 32.2
(C15), 22.1 (C10)
(40) N-acetylglycine-dihydronorcodeine HCl (2-(2-((7aR)-7-hydroxy-9-methoxy-4,4a,5,6,7,7a-hexahydro-
1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetamido)acetic acid hydrochloride) yield:
95%, m.p.: > 205 ◦C decomp., HR-MS [M + H+]: calculated: 403.1864, measured: 403.1852; 1H-NMR
(600 MHz, D2O) δ 6.87 (d, J = 8.3 Hz, H-2), 6.74 (d, J = 8.4 Hz, H-1), 4.70 (d, J = 5.4 Hz, H-5), 4.03 (d,
J = 4.7 Hz, H-6), 3.94 (s, H-9, glycine CH2), 3.76 (s, OCH3), 3.30 (d, J = 13.0 Hz, H-16), 3.12 (d, J = 19.4
Hz, H-10), 2.80–2.74 (m, CH2 bridge), 2.49 (s, H-14), 2.11 (s, H-15), 1.79 (d, J = 14.0 Hz, H-15), 1.48–1.43
(m, H-8), 0.96–0.91 (m, H-8); 13C-NMR (150 MHz, D2O)
δ 172.8 (carboxylic C=O), 165.6 (amide C=O),
145.8 (C4), 141.7 (C3), 127.9 (C12), 123.3 (C11), 120.0 (C1), 115.1 (C2), 89.2 (C5), 66.0 (C6), 61.5 (C9), 57.0
(OCH3), 54.4 (CH2 bridge), 47.9 (C16), 41.4 (glycine CH2), 40.6 (C13), 38.2 (C14), 33.9 (C15), 26.0 (C7),
21.4 (C10), 17.6 (C8)
(41) N-acetylglycine-noroxymorphone HCl (2-(2-((7aR)-4a,9-dihydroxy-7-oxo-4,4a,5,6,7,7a-hexahydro-
1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetamido)acetic acid hydrochloride) yield:
98%, m.p.: > 190 ◦C decomp., HR-MS [M + H+]: calculated: 403.1500, measured: 403.1495; 1H-NMR
(600 MHz, D2O) δ 6.73 (d, J = 8.2 Hz, H-2), 6.70 (d, J = 8.3 Hz, H-1), 4.95 (s, H-5), 4.19 (d, J = 16.1 Hz,
CH2 bridge), 3.99–3.88 (m, CH2 bridge, glycine CH2), 3.83 (d, J = 6.1 Hz, H-9), 3.33 (s, H-10), 3.20 (dd,
J = 13.2, 4.9 Hz, H-16), 3.08 (dd, J = 20.1, 6.3 Hz, H-10), 2.90 (td, J = 14.8, 4.8 Hz, H-7), 2.68 (td, J = 13.5,
5.0 Hz, H-15), 2.22 (dt, J = 14.8, 3.2 Hz, H-7), 1.99–1.92 (m, H-8), 1.72–1.59 (m, H-8, H-15); 13C-NMR
(150 MHz, D2O)
δ 211.5 (C6), 172.4 (carboxylic C=O), 165.2 (amide C=O), 143.2 (C4), 138.8 (C3), 127.1
(C12), 121.5 (C11), 121.1 (C1), 118.7 (C2), 89.3 (C5), 70.7 (C14), 65.0 (C9), 53.6 (CH2 bridge), 48.7 (C13),
47.0 (C16), 41.6 (glycine CH2), 34.5 (C7), 30.4 (C8), 27.4 (C15), 23.8 (C10)
( ) N-acetylglycine-noroxycodone HCl (2-(2-((7aR)-4a-hydroxy-9-methoxy-7-oxo-4,4a,5,6,7,7a-
42
hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-3(2H)-yl)acetamido)acetic acid hydrochloride)
◦
yield: 93%, m.p.: > 180 C decomp., HR-MS [M + H+]: calculated: 417.1656, measured: 417.1642;
1H-NMR (600 MHz, D2O)
6.89 (d, J = 8.3 Hz, H-2), 6.81 (d, J = 8.4 Hz, H-1), 4.98 (s, H-5), 4.18 (d,
J = 16.1 Hz, CH2 bridge), 3.95 (d, J = 16.2 Hz, CH2 bridge), 3.86 (d, J = 6.2 Hz, H-9), 3.79 (d, J = 3.6 Hz
δ
,
glycine CH2), 3.77 (s, OCH3), 3.35 (d, J = 20.1 Hz, H-10), 3.22 (dd, J = 13.0, 4.8 Hz, H-16), 3.11 (dd,
J = 20.1, 6.3 Hz, H-10), 2.97–2.83 (m, H-7), 2.69 (td, J = 13.5, 5.0 Hz, H-15), 2.22 (dt, J = 14.9, 3.3 Hz,
H-7), 1.98 (ddd, J = 14.5, 5.3, 3.1 Hz, H-8), 1.73–1.66 (m, H-15), 1.63 (td, J = 14.6, 3.4 Hz, H-8); 13C-NMR
(150 MHz, D2O) δ 211.3, 175.1 (carboxylic C=O), 165.2 (amide C=O), (C6), 144.0 (C4), 142.8 (C3), 127.1
(C12), 122.5 (C11), 121.0 (C1), 115.6 (C2), 89.4 (C5), 70.8 (C14), 65.0 (C9), 56.5 (OCH3), 53.8 (CH2 bridge),
48.8 (C13), 47.0 (C16), 42.8 (glycine CH2), 34.4 (C7), 30.4 (C8), 27.3 (C15), 23.8 (C10)
3.3. NMR-pH Titrations
All NMR measurements were performed on a Varian VNMRS spectrometer (599.9 MHz for 1H).
Spectra were recorded at 25 ◦C. Titrations were carried out in solutions containing 90% (v/v) H2O and
10% (v/v) D2O, with the addition of small amounts of 0.1 M HCl and NaOH. An amount of 1 M HCl
was also used to achieve highly acidic pH values. The ionic strength was kept at 0.15 M by the presence
of NaCl. The concentration of the investigated opioid compounds was 4 mM. The sample volume was
650 µL. NMR spectra were referenced to the internal standard DSS. The water signal was suppressed
by presaturation [44]. Spectra were processed using Varian VNMRj 3.2a software (Varian, Inc., NMR
Systems, Palo Alto, CA, USA). Initial pH values were read on a Metrohm 2.780.0010 precision pH meter
with a 6.0258.600 Unitrode glass Pt 1000 electrode (Metrohm AG, Herisau, Switzerland). The exact pH
in the NMR tube was measured by NMR-pH indicator molecules chloroacetic acid and dichloroacetic
acid [45] to avoid the uncertainty of the glass electrode pH-meter readings in highly acidic solutions.
The concentration of these indicators was 1.0 mM. For the analysis of NMR titration curves of proton

Molecules 2020, 25, 4009
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chemical shifts versus pH, the software Origin Pro 8 (OriginLab Corp., Northampton, MA, USA) was
used [46].
3.4. pH-Potentiometric Titrations
A 716 DMS Titrino automatic titrator (Metrohm) (Metrohm AG, Herisau, Switzerland) with
a Metrohm 6.0234.110 combined pH glass electrode was used for the potentiometric titrations,
under automatic PC (personal computer) control. The pH-potentiometric system was calibrated using
pH 1.68, 4.01, 6.87, 9.18 aqueous National Bureau of Standards (NBS) buffer solutions. Constant
temperature (25
±
0.1 ◦C) was provided by a thermostated double-walled glass cell. Difference titrations
were carried out in the absence (blank) and presence of ligands. First 2.0 mL of 0.1 M HCl solutions
were titrated with 0.1 M KOH. Constant ionic strength of 0.15 M was provided by the presence of KCl.
Then a ligand was added to the same volume of HCl solution and was subsequently titrated with
KOH. The initial concentration of the ligands varied between 1.5 and 4.0 mM in the titrations, and at
least three parallel measurements were carried out for each ligand. Non-Linear parameter fitting with
Origin 8 provided the protonation constants from the volume differences [47].
4. Conclusions
Two reaction ways have been successfully developed to obtain nitrogen linked free carboxylic group
containing morphine-derivative haptens, utilizing ethyl bromoacetate and ethyl acrylate, followed by
alkaline hydrolysis. Although the peptide bond formation was not successful, an alternative synthesis
route that used N-acetylglycine ethyl ester led to the desired glycine connected hapten derivatives.
As a result, 36 compounds were synthesized and only two of them were previously known. Detailed
NMR and HR-MS measurements confirmed the structure of all novel molecules.
In the N-acetylglycine opioid compounds, the basicity of the phenolate site is much larger than
that of the amino site because of the electron withdrawing amide group. Hydrogenation of the
C7-C8 double bond, similarly as the replacement of the electron withdrawing C6 keto group by a
hydroxyl group, increases the electron density and thus the basicity of both sites. In the N-carboxyalkyl
opioid compounds the basicity of the amino and phenolate site is within an order of magnitude in
each tribasic molecule. The higher amino basicity of the carboxylic acids compared to their ester
derivatives can be explained by the presence of a negatively charged carboxylate group in the pH
range of the amino protonation. On the other hand, the protonation state of the carboxylate group does
not significantly influence the basicity of the far-away phenolate site. The carboxylate group is less
basic in the N-carboxymethyl than in the N-carboxyethyl derivatives, because the protonated, electron
withdrawing amino group is closer to the carboxylate in the former class of compounds.
Some of these compounds are screened for opioid activity. These studies are in progress.
As continuation of this project, changing the length of the linker and coupling of other amino acids,
is planned.
Author Contributions: Conceptualization, I.K. and S.H.; methodology, I.K., B.T., K.M., G.T. and S.H.;
writing—original draft preparation, I.K., G.T., K.M. and S.H.; writing—review and editing, I.K., G.T., K.M.,
B.T. and S.H.; All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the J
Sciences (G. T th). The financial support from Bolyai + New National Excellence Program (grant number:
UNKP-19-4-SE-28) of the Ministry for Innovation and Technology is highly appreciated (G. T th). This research
ános Bolyai Research Scholarship of the Hungarian Academy of
ó
ó
was supported by the National Research, Development and Innovation Office, Hungary (NKFIH KH-130401,
VEKOP-2.3.3-15-2017-00020), the ELTE Institutional Excellence Program (1783-3/2018/FEKUTSRAT) of the
Hungarian Ministry of Human Capacities.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

Molecules 2020, 25, 4009
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