(R)-N-(1-Methyl-2-hydroxyethyl)-13-(S)-methyl-arachidonamide (AMG315): A Novel Chiral Potent Endocannabinoid Ligand with Stability to Metabolizing Enzymes

Article abstract of DOI:10.1021/acs.jmedchem.8b00611

The synthesis of potent metabolically stable endocannabinoids is challenging. Here we report a chiral arachidonoyl ethanolamide (AEA) analogue, namely, (13S,1′R)-dimethylanandamide (AMG315, 3a), a high affinity ligand for the CB1 receptor (K_i o

Full text of DOI:10.1021/acs.jmedchem.8b00611

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Article
(R)-N-(1-Methyl-2-hydroxyethyl)-13-(S)-methyl-
arachidonamide (AMG315): A Novel Chiral Potent
Endocannabinoid Ligand with Stability to Metabolizing Enzymes
Yingpeng Liu, Lipin ji, Marsha Eno, Shalley Kudalkar, Ailing Li, Marion Schimpgen, Othman Benchama,
Paula Morales, Shu Xu, Dow P. Hurst, Simiao Wu, Khadijah A Mohammad, JodiAnne T Wood,
Nikolai Zvonok, Demetris Papahatjis, Han Zhou, Chandrashekhar Honrao, Ken Mackie, Patricia
H. Reggio, Andrea Hohmann, Lawrence J. Marnett, Alexandros Makriyannis, and Spyros P. Nikas
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00611 • Publication Date (Web): 10 Sep 2018
Downloaded from http://pubs.acs.org on September 10, 2018
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(
R)-N-(1-Methyl-2-hydroxyethyl)-13-(S)-methyl-arachidonamide (AMG315): A
Novel Chiral Potent Endocannabinoid Ligand with Stability to Metabolizing
Enzymes
†,+
Yingpeng Liu, Lipin Ji, Marsha Eno, Shalley Kudalkar, Ailing Li, Marion Schimpgen, Othman
†,+
†
‡
£
#
†
§˨
‡
§
†
†
Benchama, Paula Morales, Shu Xu, Dow Hurst, Simiao Wu, Khadijah A. Mohammad, JodiAnne
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†
T. Wood, Nikolai Zvonok, Demetris P. Papahatjis, Han Zhou, Chandrashekhar Honrao, Ken
†
#
†
†
£
§
£
Mackie, Patricia Reggio, Andrea G. Hohmann, Lawrence J. Marnett, Alexandros Makriyannis,*
‡
,†,⁋
,
†
Spyros P. Nikas.*
+
equal contribution
†
Center for Drug Discovery and Department of Pharmaceutical Sciences, Northeastern University,
Boston, Massachusetts 02115, United States.
Departments of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts
2115, United States.
Departments of Biochemistry, Chemistry, and Pharmacology, Vanderbilt University School of
Medicine, Nashville, Tennessee 37232, United States.
Department of Biological and Brain Sciences, Indiana University, Bloomington, Indiana 47405,
United States.
Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vass.
Constantinou, Athens 116-35, Greece.
Center for Drug Discovery, Department of Chemistry and Biochemistry, University of North Carolina
at Greensboro, Greensboro, North Carolina 27402, United States.
⁋
0
‡
£
#
§
*
To whom correspondence should be addressed.
ABSTRACT
The synthesis of potent metabolically stable endocannabinoids is challenging. Here we report a chiral
arachidonoyl ethanolamide (AEA) analog, namely, (13S, 1'R)-dimethylanandamide (AMG315, 3a) a
high affinity ligand for the CB1 receptor (Ki of 7.8 ± 1.4 nM) that behaves as a potent CB1 agonist in
vitro (EC50 = 0.6 ± 0.2 nM). (13S, 1'R)-dimethylanandamide is the first potent AEA analog with
significant stability for all endocannabinoid hydrolyzing enzymes as well as the oxidative enzymes
COX-2. When tested in vivo using the CFA-induced inflammatory pain model 3a behaved as a more
potent analgesic when compared to endogenous AEA or its hydrolytically stable analog AM356. This
novel analog will serve as a very useful endocannabinoid probe.
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INTRODUCTION
The endocannabinoids (eCBs) N-arachidonoylethanolamine (1a, AEA, Figure 1) and 2-
arachidonoylglycerol (1b, 2-AG) are endogenously produced lipid-like substances that modulate the
1
effects of the cannabinoid receptors CB1 and CB2, two well recognized drug targets. AEA behaves as
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a low-efficacy agonist for CB1 and exhibits modest affinity for CB2. It has also been reported that
1
AEA interacts with vanilloid receptors. 2-AG is a high-efficacy agonist at both CB1 and CB2 and
appears to be the principal endocannabinoid involved in the rapid modulation of CB1/CB2 function in
1
CNS. AEA and 2-AG are short lived as they are hydrolytically deactivated by enzymes. Fatty acid
amide hydrolase (FAAH) breaks down and inactivates AEA while monoacylglycerol lipase (MGL) is
1
the major enzyme responsible for 2-AG hydrolysis. However, FAAH and a brain hydrolase (α/β-
1-2
hydrolase domain-containing 6, ABHD6) can also inactivate 2-AG. Additionally, the eCBs are
metabolized by oxygenases including cyclooxygenases (COX-2), lipooxygenases (LOXs) and
cytochrome P450 enzymes leading to eicosanoid products with distinct, non CB-mediated, biological
3
-4
actions.
COX-2 is constitutively expressed in neurons and radial glia and is upregulated in
4-5
inflammatory situations where it plays a major role in eCB metabolism. A common feature of the
major oxidative metabolic pathways of the eCBs is the abstraction of a hydrogen atom from the
3
methylene groups at C7, C10 and C13.
The four pharmacophoric regions within the structures of AEA and 2-AG (Figure 1) encompass a polar
head group, a tetraolefinic chain (TC), an n-pentyl tail and a linker which connects the head group with
6
the TC. Of these, the flexible TC is the major determinant of the biologically active conformation(s) of
6-9
the eCBs as reflected in their binding motifs and G-protein signaling properties. A key structural
feature of the TC is the bis-allylic carbons (7, 10 and 13), that are the carbons connected to two
consecutive C=C double bonds. These methylene groups can access a broad region of conformational
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space with the TCs exhibiting a high degree of flexibility. This situation permits the arachidonoyl
moiety of the lipid to assume a number of conformations including both folded as well as extended
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conformations. Studies on AEA and its analogs have suggested that the folded U-, J-, or helical-shaped
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conformations are responsible for CB1 receptor recognition
and successful modifications at the
polar head, linker and tail groups of the AEA have been reported with the best-known being the (R)-
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, 12-16
methanandamide (AM356),
a valuable AEA analog which exhibits enhanced stability for FAAH.
However, ethanolamides with structurally modified or constrained TCs that mimic the above types of
conformations are either inactive, or exhibit much lower affinities for the CB1 receptor when compared
8
, 11, 15, 17
to AEA.
Worthy of note, ethanolamides with significant affinities for CB2 have not been
reported, and there are no reports on AEA analogs that are stable with the major oxidative enzyme
7, 18
COX-2. Recently, we reported the crystal structures of the CB1 receptor.
This breakthrough work in
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9-20
combination with biophysical methods,
computational studies and currently available biochemical
and in vivo assays opens the door to explore the molecular basis regarding the bioactive eCB
conformation(s) as well as downstream signaling events that are associated with the in vitro and in vivo
pharmacological profiles of these lipids. Taking into consideration all the above, we report here the
discovery of novel chiral AEA probes that encompass TCs with distinct conformational properties,
higher binding affinities for CB1 and CB2 receptors, increased abilities to activate their molecular
targets (CB1, CB2), and enhanced stabilities for their deactivating enzymes FAAH and COX-2. Our
lead analog, namely, (13S, 1'R)-dimethyl-anandamide (3a) is a novel chiral eCB ligand possessing high
affinity and potency for the CB1 receptor coupled with excellent biochemical stability for FAAH,
MGL, ABHD6 and COX-2. This analog suppressed CFA-induced mechanical allodynia through a CB1
mechanism and produced greater anti-allodynic efficacy than its hydrolytically stable analog AM356.
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RESULTS AND DISCUSSION
Ligand Design
A very useful approach for the design of the novel AEA analogs with variable conformational and
stability properties is the addition of chiral methyl groups in each of the three C7, C10 and C13 bis-
allylic carbons of AEA (Figure 2). The value of this approach was initially recognized by our
preliminary finding that addition of a methyl group at the 13S position of AEA led to an enhanced
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affinity for the CB1 receptor. Our first structure-activity/-stability study in which we introduced chiral
methyl groups in the bis-allylic carbons of AEA led to chiral Me-arachidonoyl ethanolamides (chiral-
Me-AEAs, 2). We showed that when methyl groups with preferred chiralities were introduced at the C7
or C13 positions enhanced the affinities of these analogs for CB1 and CB2 while the C10 methyl
analogs were inactive. Subsequently, the most promising analogs (C13- and C7-chiral-Me-AEAs) were
modified at the polar head and tail groups to further probe CB receptor recognition in tandem with
increasing the stability of the amide group for FAAH. This was pursued through two design
approaches. First, we preserved the recognition features of the endogenous ethanolamide head and, as
15, 21
in our earlier work,
we added a second chiral methyl alpha to the nitrogen atom. Second, we
replaced the ethanolamine group of our lead (13S)-Me-AEA (2a) with amine groups, including bulky
amines, that lack the hydroxyl group of the prototype. These design approaches led to chiral di-Me-
arachidonoyl ethanolamides (chiral-di-Me-AEAs, 3) and (13S)-Me-arachidonoyl amides [(13S)-Me-
AAs, 4].
Chemistry
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, 17, 22
Based on our preliminary studies,
our optimized synthetic strategy involves key methyl ester
precursors (5, Figure 3) from which the chiral AEA probes are produced through peptide coupling. The
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retrosynthetic analysis disconnects each compound at the double bonds adjacent to the chiral centers by
retro-Wittig-type reactions leading to the chiral aldehyde fragments, the head group fragments, and the
tail fragments. The strategy was successfully executed after securing these fragments in
stereochemically pure form by acetylene alkylation and selective P-2 nickel hydrogenation. Notable is
the robustness of the optimized multi-step enantioselective syntheses that allow the synthesis of the
final compounds in substantial quantities as needed for further in vitro and in vivo studies.
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Synthesis of the chiral aldehydes 8a and 8b was carried out using a modification of our earlier reported
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procedure that improved the overall yields and reduced the synthetic steps (Scheme 1). Thus,
silylation of commercially available methyl esters 6a and 6b gave the respective TBDPS ethers 7a and
7b in excellent yields (97-98%). The subsequent reduction with diisobutylaluminum hydride took place
o
at -110 C using 1.2 equivalents of the DIBAL-H reagent and led to aldehydes 8a and 8b in high yields
(87-88% after purification) while the formation of the overreduction byproducts alcohols was
substantially suppressed (3-5%).
Synthesis of the alkenyl phosphonium salt 14 which is required for the construction of the chiral C7-Me
AEA analogs is depicted in Scheme 2. Copper mediated cross-coupling of 3-butyn-1-ol (9) with
commercially available bromide 10 afforded diyne alcohol 11 in 93% yield. Partial hydrogenation of 11
6
, 22
over P-2 nickel catalyst gave the corresponding skipped Z diene alcohol 12 in 96% yield.
Reaction
of triphenylphosphine and carbon tetrabromide with alcohol 12 under mild conditions produced
o
bromide 13 (94% yield). Heating 13 (70-75 C) with dry PPh under an argon atmosphere for five days
3
6
, 22
afforded the phosphonium salt 14 in 90% yield.
The four protons of the two double bonds in
1
3
3
compound 14 are well separated in the H NMR spectrum and the coupling constants J - and J -
3
H 4H
5H
were found to be less than 10.7 Hz. This confirms a Z stereochemistry between the hydrogens in the
H
6
3H-4H and 6H-7H spin systems.
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The assembly of the triphenylphosphonium bromides 14 and 15 (commercially available) with chiral
aldehydes 8a and 8b into (7R)- and (7S)-Me-AEAs 2e and 2f, respectively, is outlined in Scheme 3.
Thus, exposure of 15 to potassium bis(trimethylsilyl)amide at room temperature and reaction of the
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resulting ylide with aldehydes 8a and 8b at -115 C gave the carboxylic acid derivatives 16a and 16b,
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respectively (73-75% yields). Based on H NMR data this Wittig reaction gave exclusively the Z
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alkenes with J - ~ 10.7 Hz. Exposure of 16a and 16b to TMSCHN in Et O/MeOH esterified the
5H 6H
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carboxylic acid group leading to methyl esters 17a and 17b, respectively.
This was followed by
removal of the TBDPS group using TBAF to give 18a and 18b in 73-75% yields for the two steps.
Dess-Martin periodinane oxidation of alcohols 18a and 18b led to aldehydes 19a and 19b, respectively.
Due to their labile character these intermediates were used immediately in the subsequent Wittig
olefination reaction with phosphonium salt 14 to give the respective (7R)- and (7S)-methyl
arachidonates 20a and 20b in very good yields (69-71%) from alcohols 18a and 18b. In agreement with
our earlier work on similar systems as well as prostaglandin analogs this final Wittig olefination
6
, 17, 22-23
reaction is stereoselective and installs the C8=C9 double bond with the Z stereochemistry.
Saponification of 20a and 20b (LiOH in THF/H O) afforded acids 21a and 21b (94-96% yields), that
2
were coupled with 2-(tert-butyldiphenylsilyloxy)ethanamide 23 to afford the silylated amide precursors
2
4a and 24b in excellent yields (95-97%) by using the N,N'-carbonyldiimidazole-mediated amide
17, 22
coupling.
Deprotection of the tert-butyldimethylsilyl ether was carried out by using TBAF in THF
and gave the enantiomeric (7R)- and (7S)-methyl anandamides 2e and 2f, respectively, in 85-88%
yields.
The enantioselective syntheses of (10S)-, (10R)-, and (13S)-methyl-anandamides 2c, 2d and 2a,
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, 22
respectively, (Figure 2) were reported earlier from our group.
The synthesis of the (13R)-methyl-
anandamide 2b is summarized in Scheme 4 and starts from the phosphonium salt 25. This advanced
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key intermediate is common for the synthesis of all (13S)- and (13R)-methyl substituted AEA analogs
and it was synthesized in multigram quantities in eight steps starting from commercially available 3-
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butyn-1-ol as we described earlier. Wittig olefination of aldehyde 8b with 25 at low temperature, in the
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presence of potassium bis(trimethylsilyl)amide, produced exclusively the desired Z-alkene product 26
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in excellent yield (91%, J
-
1H 12H
= 10.8 Hz). This was followed by fluorodesilylation employing TBAF
1
in THF and gave alcohol 27 in 91% yield. Mild oxidation of 27 with the Dess-Martin periodinane
reagent led to aldehyde 28 which was used immediately in the next step without purification. The
Wittig olefination reaction of 28 with the ylide derived from hexyltriphenylphosphonium bromide and
potassium bis(trimethylsilyl)amide worked well and produced methyl ester 29 in 65% yield from
alcohol 27. As expected, the Wittig reaction produced exclusively the desired Z geometrical isomer
under the experimental conditions used (low temperature, salt free). This is in full agreement with our
earlier work on the (13S)-enantiomer (2a) where the Z stereochemistry of the C14=C15 double bond
6
was unambiguously confirmed by using a combination of 1D and 2D NMR methods. Subsequently,
saponification (85% yield), CDI mediated coupling with protected ethanolamide 23 (93% yield), and
fluorodesilylation with TBAF (89% yield) led to target (13R)-Me-AEA (2b).
The chiral di-Me-AEA analogs 3a-e were synthesized in a similar manner as depicted in Scheme 5.
6
Thus, Wittig olefination of aldehyde 8a with 25 followed by fluorodesilylation gave the Z alcohol 31.
The synthesis of the requisite tail modified acid 34b was accomplished by following: a) oxidation with
Dess-Martin periodinane, b) Wittig olefination, and c) methyl ester hydrolysis. Eventually, amide
coupling of the acids 34a-e with the chiral protected ethanolamine 36 produced the intermediate
silylated amides 37a-e that upon treatment with TBAF afforded the chiral di-Me-AEA analogs 3a-e.
Varying the head moieties of the (13S)-Me-substituted arachidonoyl skeleton begins with the free acid
34a coupling to the amine derivatives to give the desired amide analogs 4a-e (Scheme 6). Again, the
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CDI activation procedure worked well and produced these analogs in excellent yields (85-92%).
Modifications at the tail aimed at incorporating azido and isothiocyanato functionalities are shown in
Scheme 6. The requisite tail fragment 40 was synthesized in two steps from commercially available 6-
bromo-hexan-1-ol (38) by using TBDPS protection and phosphonium salt formation reactions. Starting
from alcohol 31 the Dess-Martin periodinane oxidation-Wittig olefination led to the intermediate
methyl ester 41 in 64% overall yield. The terminal hydroxy group was then deprotected using TBAF to
give 42 in 86% yield. At this point we had established a path to synthesize the key primary alcohol 42
which allows the further functionalization of both the terminal tail carbon and the polar head group. In
the current study we focused on probes incorporating azido and isothiocyanato moieties in analogs
bearing the cyclopropylamido head group. Thus, heating 42 with diphenylphosphoryl azide and DBU
in dry DMF gave azide 43 in 71% yield. Saponification of azide ester 43 with LiOH led to acid 44
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(95% yield) which was subjected to CDI-mediated amide coupling with cyclopropyl amine to give
amide 4f (74% yield). Subsequently, the isothiocyanato probe 4g was isolated after treatment of the
azido probe 4f with carbon disulfide in the presence of triphenylphosphine (96% yield).
Biochemical characterization
The synthesized chiral AEA analogs were evaluated in four steps to assess their in vitro binding
affinities, functional properties and stability for FAAH and COX-2, as follows: 1) all analogs were
assessed for affinity binding to rCB1, mCB2 and hCB2; 2) the most promising compounds from each
series were further evaluated in the adenylyl cyclase (cAMP) assay as a measure of their functional in
vitro potency and efficacy at the CB1 receptors; 3) all analogs were evaluated preliminarily for their
stability towards FAAH by comparing the Ki values from the rCB1 binding assays in the presence and
absence of the FAAH inhibitor phenylmethanesulfonylfluoride (PMSF) as we have carried out in the
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past (detailed below).
This screening process served as a guide to select key AEA probes for
further studies where their stabilities for expressed human and rat FAAH were assessed precisely using
LC-MS/MS; 4) representative analogs were tested for their stability towards COX-2. The discussion of
the biochemical characterization follows.
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a) In vitro biological activity
All AEA analogs (series 2, 3, and 4) were assessed for affinity binding to rCB1, mCB2 and hCB2
3
receptors. Competition binding experiments using [ H]CP-55,940 as the radioligand were performed in
6
rat brain or HEK293 cell membrane preparations. As we detailed elsewhere, amidase activity is
present in rCB1 receptor preparations. Thus, binding experiments were carried out with and without
pretreatment of the rCB1 membranes with phenylmethanesulfonyl fluoride (PMSF), a common
protease inhibitor. CB2 affinity was measured by using receptor preparations where FAAH activity is
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absent. The choice of the widely used radioligand [ H]CP-55,940 as a competing ligand for the
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binding assays was based on considerations discussed in our earlier publications.
Moreover, as we
detailed in earlier studies, the CB2 receptor shows less homology (~82%) between species than does
CB1 (~97-99%), and this may result in species-related differences in affinity. Therefore, the AEA
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analogs were tested on both, mouse and human CB2. Functional characterization of key AEA analogs
6
possessing the highest affinities for the rCB1 receptor was performed by measuring changes in the
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forskolin-stimulated cAMP as we reported earlier.
An examination of the binding affinity data of the chiral Me-arachidonoyl ethanolamides (chiral-Me-
AEAs, 2, Table 1) shows that presence of a chiral methyl group at the tetraolefinic chain (TC) has a
striking effect on the binding affinity and selectivity of the analogs for CB1 and CB2. Thus, addition of
a (13S)-methyl group, compound 2a, results in a remarkable increase in the affinity of the analog for
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the CB1 receptor when compared to endogenous ligand AEA. However, the (13R)-enantiomer 2b is
inactive at both CB1 and CB2 receptors. In contrast, both the (10S) and the (10R) stereochemistry is
inactive at CB1 and CB2 as seen by comparing the binding affinities of the enantiomers 2c and 2d
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(Table 1). The effect of the chiral methyl group at C7 on the binding affinity of the compounds is more
complex. Thus, the (7R)-enantiomer 2e is a high affinity CB1/CB2 ligand while the (7S)-enantiomer 2f
has low affinity for both the CB1 and CB2 receptors. Cyclase data (Table 2) confirmed the effect of the
stereochemistry at C7 and C13 on the bioactivity of these analogs and showed that the AEA analogs 2a
and 2e behave as potent CB1 agonists.
Binding affinities and cyclase data for the chiral-di-Me-AEA analogs 3 are summarized in Tables 3 and
4. Our data show that addition of a second chiral methyl group at C1' of the enantiomers 2a and 2b lead
to diastereomeric counterparts 3a and 3cb, respectively, with similar SAR trends (Table 3). Thus,
analog 3a with the 13S stereochemistry is a high affinity ligand for rCB1 while again, the 13R
stereochemistry is not recognized by either the CB1 or the CB2 receptors (analog 3c). Comparisons of
the binding affinities of the chiral analogs 2a and 3a with their achiral congeners AEA and AM356,
respectively (Tables 1 and 3), confirm the superior cannabinergic activity of the chiral 13S-Me-AA
chain versus the non-chiral AA-chain. As seen in analog 3b, transformation of the linear n-pentyl tail of
the 13S,1'R-di-Me-AEA 3a to the longer and bulkier phenoxypropyl group results in a reduction of the
affinity of the analog for the CB receptors which is more accentuated in CB1. Incorporation of a second
chiral methyl group at the C1' of the C7-chiral-Me-AEA analogs 2e and 2f leads to diastereomers 3d
and 3e (Table 3), respectively, that are both high affinity ligands for CB1 and CB2 receptors. This
suggests that the C7 position is more tolerant to the structural changes when compared to C13. Our
functional characterization study found that the key chiral-di-Me-AEA analogs 3a, 3d and 3e potently
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decrease the levels of cAMP, indicating that within this signaling mechanism these compounds behave
as potent agonists at CB1 (Table 4).
The Ki values for the (13S)-Me-arachidonoyl amides 4 are presented in Table 5 and the functional
potencies of representative analogs in Table 6. We observe that the cyclopropyl (4a), 2'-chloroethyl
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(4d) and propargyl (4e) analogs have the highest binding affinities for CB1, approximately 17-70-fold
higher than the endogenous AEA while key representative analogs (4a, 4b, 4d and 4f) behave as potent
agonists for CB1 in the cAMP assay. A cursory examination of the binding affinities of the analogs 4a,
4
b and 4c for CB1 suggest that increasing the size of the cycloalkylamino head group from a
cyclopropyl to a bulkier cyclobutyl or cyclopentyl ring leads to a progressive reduction of the affinity
for CB1. Interestingly, analogs 4a, 4b, 4d and 4e exhibit substantial binding affinity for the CB2
receptor with preference for the hCB2 versus the mCB2. To this end the cyclobutyl analog 4b has a 3-
1
3-fold preference for hCB2 over rCB1 and mCB2. Incorporation of a polar azido or isothiocyanato
group at the terminal carbon atom (C20) of the chiral cyclopropylamido analog 4a leads to head/tail
hybrid analogs 4f and 4g that bind equally well to both rCB1, mCB2 and hCB2 receptors. This supports
a more general observation through this SAR study, that combinations of the TC’s chiral features with
appropriate head- and/or tail-groups can generate AEA analogs with high affinity for CB2, a finding
that has not been reported for AEA analogs. Additionally, analogs 4f and 4g carrying the
photoactivatable azido and the electrophilic isothiocyanato groups were tested for their ability to
irreversibly label the rCB1 and the hCB2 receptors by following established procedures developed in
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our laboratory.
Our studies indicated that both 4f and 4g are not covalent binders at CB1 and
CB2. Overall, our biochemical evaluation led to the discovery of novel chiral AEA ligands with distinct
biological profiles including CB1 selective (e. g., 2a and 3a), and dual CB1/CB2 probes (e.g., 2e, 3d,
4a, 4b, 4f and 4g). Key representative analogs behave as potent CB1 agonists.
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b) Metabolic stability for FAAH and COX-2
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Metabolic stability for AEA and representative chiral-Me-AEAs, chiral-di-Me-AEAs and (13S)-Me-
AAs are summarized in Table 7. As we reported in earlier studies, comparisons of a compound’s Ki
values for rCB1 obtained with and without the presence of PMSF (see Tables 1, 2 and 4), can serve as a
preliminary assessment of the stability of that compound towards FAAH hydrolysis. Thus, the rCB1
binding assays were performed twice for each AEA analog, once after treatment of the membranes with
PMSF to protect against FAAH hydrolysis, and a second time without pretreating the membranes with
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PMSF where enzymatic activity is not blocked.
Very little or no effect of PMSF on CB1 receptor
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binding affinity (Ki) is considered as an indication of enzymatic stability (see Table 7).
This
preliminary screening of AEA and its analogs served as a guide to select AEA probes for further
studies where their stabilities for expressed human FAAH were assessed quantitatively using LC-
MS/MS methods. For species comparisons (rat versus human) AEA and one of the chiral analogs (3d)
were tested in both rat and human FAAH preparations. Overall, our studies have identified AEA probes
with excellent stability for the human FAAH enzyme including all chiral-di-Me-AEA ligands (3) and
the (13S)-Me-AA analogs 4b, 4c, 4d, 4f, and 4g. In addition, our data indicate that AEA is recognized
equally well from both the rat and human clone while 3d maintains its excellent FAAH stability for
31
both species. As in earlier work, we have used a polarographic assay that measures oxygen
consumption to investigate the endogenous AEA, arachidonic acid, and key analogs as substrates for
mCOX-2 activity. Data are presented in Figure 4. In agreement with our rational design our studies
indicate that incorporation of a chiral methyl group at positions C7 and C13 of the endogenous
prototype enhances the stability of the TCs towards COX-2. A finding which is reported here for first
time for AEA ligands. Our key analog 3a [(13S, 1'R)-di-Me-AEA] was further tested for stability in
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both human MGL and human ABHD6 using HPLC methods (see experimental section) and found to be
stable for 30 min.
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Molecular modeling
In an effort to understand the large binding affinity difference between the two AEA analogs, 3a and 3c
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we performed docking studies in the CB R* model as we reported in earlier work. Methods are
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detailed under Supporting Information. As illustrated in Figure 5A, our docking studies suggest that 3a
sits high in the receptor in the TMH2-3-6-7 region and establishes its primary interaction with K3.28.
The (13S)-methyl group of 3a defines its particular binding affinity and makes it enantioselective at
CB . This stereocenter adds conformational restriction in this region of the molecule directing the
1
methyl group into a hydrophobic pocket defined by K3.28, L3.29 and above V3.32, establishing strong
Van der Waals interactions. The alkyl tail is directed upward in the binding site, establishing
hydrophobic interactions with T3.33 and Y5.39. Attempts were made to dock 3c in the same binding
pocket region as the 3a binding site. However, the (13R)-methyl group of 3c forces the ligand towards
TMH6 where it has steric overlap with L6.51 (Figure 5B). This overlap precludes 3c binding at CB1.
Our docking studies provide a rational structural basis of the CB enantioselectivity of 3a over 3c.
1
Although the enantioselective actions of THC and related exogenous cannabinoids on CB1 receptor are
well established, this phenomenon is observed here for first time with the endocannabinoid scaffold.
In vivo behavioral characterization
a) Results. AMG315 produced anti-allodynic effect in CFA-induced inflammatory pain which was CB1
receptor dependent. We first examined the anti-allodynic efficacy of AEA (20 mg/kg i.p.) in CFA-
induced inflammatory pain. Intraplantar injection of CFA significantly reduced the mechanical paw
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withdrawal threshold on the ipsilateral side (injected paw) (F 1, 10 = 268.2, p < 0.001) to similar extent
in both groups (F 1, 10 = 0.120, p = 0.736) before pharmacological treatments (Figure 6A), no group
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differences in inflammatory nociception was observed prior to pharmacological manipulations (F 1, 10
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.016, p = 0.903). During the maintenance phase of CFA-induced inflammatory pain, mechanical
thresholds changed over time (F 3, 30 = 7.161, p < 0.001) but did not differ between groups (F 1, 10
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.717, p = 0.219), although the interaction (F 3, 30 = 3.563, p = 0.026) between groups and time were
significant (Figure 6A). Bonferroni post hoc tests revealed that AEA produced only a transient anti-
allodynic effect at 10 minutes post injection (p = 0.014) and anti-allodynic efficacy was no longer
present at 30 minutes post injection (Figure 6A). This transient anti-allodynic effect of AEA is
consistent with rapid degradation of this fatty-acid amide by fatty acid amide hydrolase (FAAH).
We then compared the anti-allodynic effects of compounds likely to exhibit improved metabolic
stability compared to AEA. We compared the antiallodynic efficacy of AMG315 (5 mg/kg i.p.) and
AM356 (20 mg/kg i.p.) in the same pain model (Figure 6C). During the maintenance phase of CFA-
induced inflammatory pain, AMG315 (5 mg/kg, i.p.) and AM356 (20 mg/kg, i.p.) increased mechanical
paw withdrawal thresholds (F 2, 15 = 20.82, p < 0.001) in a treatment-dependent manner (F 6, 45 = 4.706,
p = 0.001) and the mechanical paw withdrawal threshold changed over time (F 3, 45 = 16.1, p < 0.001)
(Figure 6C). Bonferroni post hoc tests revealed that both AMG315 (5 mg/kg) and AM356 (20 mg/kg)
significantly increased mechanical paw withdrawal threshold at 10 minutes post injection compared
with the vehicle group (p < 0.001) (Figure 6C). Moreover, mechanical paw withdrawal threshold was
higher in CFA-injected mice that received AMG315 (5 mg/kg i.p.) compared to AM356 (20 mg/kg) at
1
0 minutes post injection (p = 0.021), consistent with improved efficacy of AM315 compared to
AM356. In contrast to AEA, both AMG315 and AM356 displayed longer duration of action which
lasted up to 30 minutes (Figure 6C). In addition, only AM356 (20 mg/kg) increased mechanical paw
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withdrawal thresholds in the CFA-injected paw compared to mice receiving vehicle at 60 minutes post
injection (p = 0.014). Mechanical paw withdrawal threshold in the CFA-injected paw were similar in
mice receiving AM315 and AM356 at this time point (p = 1).
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To determine if anti-allodynic effects of AMG315 were mediated by CB1 receptors, the antiallodynic
effect of AMG315 (5 mg/kg, i.p.) was compared in WT and CB1 KO mice (Figure 6E). Post-injection
mechanical paw withdrawal thresholds differed between groups and across post-injections times and
the interaction between group and time was significant (F 2, 15 = 17.545, p <0.001 [group]; F 3, 45 = 9.67,
p < 0.001 [time]; F 6, 45 = 2.961, p = 0.016 [interaction]) (Figure 6E). WT mice receiving AMG315
showed elevated mechanical paw withdrawal thresholds compared to the vehicle group at 10 and 30
minutes post injection (p < 0.001). By contrast, mechanical paw withdrawal thresholds in CB1 KO
mice receiving AM315 did not differ from that of the vehicle group at any time points tested (Figure
6
E). The CFA injection induced a slight increase in the mechanical paw withdrawal threshold on the
contralateral side (un-injected paw), an expected consequence of uneven weight bearing of the hind
paws after CFA injection (Figure 6, D, F). However, pharmacological manipulations did not alter the
mechanical paw withdrawal threshold of the contralateral paw during the maintenance phase of CFA-
induced inflammatory pain at any timepoint (Figure 6B: F 3, 30 = 1.016, p = 0.400 [time]; F 1, 10 = 0.024,
p = 0.880 [group]; F 3, 30 = 1.134, p = 0.351 [interaction]; Figure 6D: F 3, 45 = 0.515, p = 0.674 [time]; F
=
0.616, p = 0.553 [group]; F 6, 45 = 0.189, p = 0.978 [interaction]; Figure 6F: F 3, 45 = 0.328, p =
.850 [time]; F 2, 15 = 0.835, p = 0.453 [group]; F 6, 45 = 0.499, p = 0.806 [interaction]).
2
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b) Discussion. The metabolically stable anandamide analog AMG315 (5 mg/kg i.p.) suppressed CFA-
induced mechanical allodynia through a CB1 mechanism. Antinociceptive effects of AMG315 were
restricted to the CFA-injected (ipsilateral) paw and were absent in the noninjected (contralateral) paw.
The duration of action of AMG315 and AM356 outlasted that of AEA. AMG315 also produced greater
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anti-allodynic efficacy than AM356 at 10 minutes post injection, but did not exhibit a longer duration
of action than AM356, suggesting possible superiority of AMG315 relative to AM356. By sparing
metabolic degradation by both FAAH and COX, the magnitude of anti-allodynic efficacy may be
increased preferentially (as opposed to duration of action). The anti-allodynic efficacy of AMG315
observed in WT mice was largely absent in CB1KO mice, suggesting a CB1 mechanism of action. The
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same vehicle alone did not alter the responding in CB1 KO mice in our previous work.
CONCLUSIONS
We report the first AEA probe, namely, (13S, 1'R)-dimethyl-anandamide [(13S, 1'R)-di-Me-AEA, 3a]
possessing high affinity and potency for the cannabinoid CB1 receptor coupled with excellent
biochemical stability for FAAH, MGL, ABHD6 and COX-2. When tested in vivo using the CFA-
induced inflammatory pain model 3a behaved as a more potent analgesic when compared to
endogenous AEA or its hydrolytically stable analog AM356.
This was the result of a careful SAR study that led to novel AEA ligands with modified tetraolefinic
chains and unique pharmacological profiles including CB1 selective (e. g., 2a and 3a), and dual
CB1/CB2 probes (e.g., 2e, 3d, 4a, 4b, 4f and 4g). Our approach for developing the novel metabolically
stable AEA analogs was based on introducing chirality with conformational restriction in the
tetraolefinic chain of the prototype. Key representative analogs behave as potent CB1 agonists and have
enhanced stability for COX-2 while the head group analogs are endowed with stability towards FAAH.
Unlike AEA the chiral ligands reported here do not generate arachidonic acid which exhibits non-CB-
mediated biological actions as well as in vitro and in vivo toxicity. As such, these ligands will be
valuable experimental tools currently lacking for future in vitro, in vivo, and theoretical studies aimed at
exploring the (patho)physiological roles of the endocannabinoid system.
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EXPERIMENTAL SECTION
Materials: All reagents and solvents were purchased from Aldrich Chemical Company, unless
otherwise specified, and used without further purification. All anhydrous reactions were performed
under a static argon or nitrogen atmosphere in flame-dried glassware using scrupulously dry solvents.
Flash column chromatography employed silica gel 60 (230-400 mesh). All compounds were
demonstrated to be homogeneous by analytical TLC on pre-coated silica gel TLC plates (Merck, 60
F245 on glass, layer thickness 250 ꢀm), and chromatograms were visualized by phosphomolybdic acid
staining. Melting points were determined on a micro-melting point apparatus and are uncorrected.
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1 13
NMR spectra were recorded in CDCl , unless otherwise stated, on Varian 600 ( H at 600 MHz, C at
3
1
50 MHz), Varian 500 ( H at 500 MHz), Varian 300 ( C at 75 MHz), and Bruker 400 ( H at 400 MHz,
13
1
1
13
C at 100 MHz) NMR spectrometers and chemical shifts are reported in units of
TMS. Multiplicities are indicated as br (broadened), s (singlet), d (doublet), t (triplet), q (quartet), m
multiplet) and coupling constants (J) are reported in hertz (Hz). Low and high-resolution mass spectra
δ
relative to internal
(
were performed in School of Chemical Sciences, University of Illinois at Urbana-Champaign. Mass
spectral data are reported in the form of m/z (intensity relative to base = 100). Purities of the tested
compounds were determined by LC/MS analysis using a Waters MicroMass ZQ system (electrospray
ionization (ESI) with Waters-2525 binary gradient module coupled to a photodiode array detector
(Waters-2996) and ELS detector (Waters-2424) using a XTerra MS C18 (5 µm, 4.6 mm × 50 mm
column and acetonitrile/water) and were > 95 %.
(13R,5Z,8Z,11Z,14Z)-N-(2-Hydroxyethyl)-13-methyleicosa-5,8,11,14-tetraenamide (2b). To a
solution of 30 (25 mg, 0.04 mmol) in dry THF (0.8 mL) at 0 ºC, under an argon atmosphere, was added
tetra-n-butylammonium fluoride (TBAF, 0.05 mL, 0.05 mmol, 1M solution in THF) dropwise. The
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reaction mixture was stirred for 10 min at 0 ºC and for 1.5 hours at room temperature, and then it was
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quenched with saturated aqueous NH Cl at 0 ºC and extracted with EtOAc. The combined organic
4
extracts were washed with brine, dried over MgSO and the solvent was evaporated under reduced
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pressure at 35-37 ºC. The residue was purified by flash column chromatography on silica gel (40 %
1
EtOAc/hexane) to give 2b (13 mg, 89 % yield) as a colorless oil. H NMR (500 MHz, CDCl ) δ 0.89 (t,
3
J = 6.9 Hz, 3H, 20-H), 1.02 (d, J = 7.2 Hz, 3H, >CH-CH ), 1.24-1.33 (m, 4H, 18-H, 19-H), 1.35
3
(
sextet, J = 7.2 Hz, 2H, 17-H), 1.73 (quintet, J = 7.2 Hz, 2H, 3-H), 2.06 (nonet, J = 6.3 Hz, 2H, 16-H),
.12 (dt, J = 7.2 Hz, J = 7.2 Hz, 2H, 4-H), 2.22 (t, J = 7.6 Hz, 2H, 2-H), 2.52 (br s, 1H, -OH), 2.76-2.84
ddd and t, overlapping, 3H, 10-H and 7-H, especially 2.81, t, J = 6.0 Hz, 2H, 7-H), 2.87 (ddd, J = 15.0
Hz, J = 7.5 Hz, J = 6.3 Hz, 1H, 10-H), 3.42 (dt, J = 5.2 Hz, J = 5.2 Hz, 2H, -CH -N<), 3.46 (sextet, J =
2
(
2
7
.5 Hz, 1H, 13-H), 3.73 (t, J = 5.2 Hz, 2H, -CH O-), 5.20-5.31 (m, 4H, 11-H, 12-H, 14-H, 15-H), 5.44-
2
1
3
5
.32 (m, 4H, 5-H, 6-H, 8-H, 9-H), 5.88 (br s, 1H, >NH); C NMR (150 MHz, CDCl ) δ 174.16
3
(>C=O), 135.10 (C-12), 134.12 (C-14), 129.02 (C-8), 128.84 (C-5), 128.43 (C-6), 128.26 (C-15),
1
1
2
28.10 (C-9), 125.61 (C-11), 62.70 (-CH -OH), 42.48 (NH-CH ), 35.92 (C-2), 31.57 (C-18), 30.50 (C-
2 2
3), 29.46 (C-17), 27.51 (C-16), 26.61 (C-4), 25.84 (C-10), 25.61 (C-7), 25.44 (C-3), 22.59 (C-19),
+
2.04 (C-13-CH ), 14.09 (C-20). Mass spectrum (ESI) m/z (relative intensity) 384 (M + Na), 362 (M
+
3
+
H, 100), 301 (7). Exact mass (ESI) calculated for C H NO (M + H), 362.3059; found, 362.3047.
+
2
3
40
2
LC/MS analysis (Waters MicroMass ZQ system) showed purity 98% and retention time 5.8 min for the
title compound.
(7R,5Z,8Z,11Z,14Z)-N-(2-Hydroxyethyl)-7-methyleicosa-5,8,11,14-tetraenamide
(2e).
The
synthesis was carried out as described for 2b bellow, using 24a (30 mg, 0.05 mmol) and TBAF (0.07
mL, 1M solution in THF) in dry THF (3 mL). The reaction was completed in 1 hour and the crude oil
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obtained after work up was purified by flash column chromatography on silica gel (15%
EtOAc/hexane) to give 2e (15 mg, 85 % yield) as a colorless oil. IR (neat): 3303, 3012, 2927, 2857,
-
1 1
1646 (C=O), 1543, 1275, 1261, 1066 cm ; H NMR (500 MHz, CDCl ) δ 0.89 (t, J = 6.8 Hz, 3H, 20-
3
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H), 1.02 (d, J = 6.8 Hz, 3H, >CH-CH ), 1.23 – 1.41 (m, 6H, 17-H, 18-H, 19-H), 1.73 (quintet, J = 7.5
3
Hz, 2H, 3-H), 2.06 (dt, J = 7.3 Hz, J = 7.3 Hz, 2H, 16-H), 2.09 – 2.18 (m, 2H, 4-H), 2.21 (t, J = 7.5 Hz,
2
H, 2-H), 2.73 – 2.94 (m, 4H, 10-H, 13-H), 3.38 – 3.50 (sextet and dt overlapping, 3H, 7-H, -CH -NH-
2
), 3.72 (t, J = 5.0 Hz, 2H, -CH OH), 5.20 – 5.46 (m, 8H, 5-H, 6-H, 8-H, 9-H, 11-H, 12-H, 14-H, 15-H),
2
13
.87 (br s, 1H, -NH-). C NMR (100 MHz, CDCl ) δ 174.1 (C=O), 135.3 (C=C), 134.7 (C=C), 130.5
5
3
(C=C), 128.6 (C=C), 128.0 (C=C), 127.5 (C=C), 126.8 (C=C), 125.9 (C=C), 62.7 (CH OH), 42.5 (-
2
NHCH -), 36.0, 31.5, 30.5, 29.3, 27.2, 26.9, 25.8, 25.6, 25.5, 22.5, 22.0, 14.0. Mass spectrum (ESI) m/z
2
+
+
relative intensity) 362 (M + H, 100). Exact mass (ESI) calculated for C H NO (M + H), 362.3059;
(
23 40 2
found, 362.3073. LC/MS analysis (Waters MicroMass ZQ system) showed purity 99% and retention
time 5.4 min for the title compound.
(7S,5Z,8Z,11Z,14Z)-N-(2-Hydroxyethyl)-7-methyleicosa-5,8,11,14-tetraenamide
(2f).
The
synthesis was carried out as described for 2b bellow, using 24b (30 mg, 0.05 mmol) and TBAF (0.07
mL, 1M solution in THF) in dry THF (3 mL). The reaction was completed in 1 hour and the crude oil
obtained after work up was purified by flash column chromatography on silica gel (15 %
1
EtOAc/hexane) to give 2f (16 mg, 88 % yield) as a colorless oil. The IR, H NMR (500 MHz, CDCl )
3
1
3
and C NMR (100 MHz, CDCl ) spectrum was identical to that of the enantiomer 2e. Mass spectrum
3
+
ESI) m/z (relative intensity) 384 (M + Na, 18), 362 (M + H, 100), 301 (7). Exact mass (ESI)
+
(
+
calculated for C H NO (M + H), 362.3059; found, 362.3062. LC/MS analysis (Waters MicroMass
2
3
40
2
ZQ system) showed purity 99 % and retention time 5.4 min for the title compound.
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(13S,5Z,8Z,11Z,14Z)-N-((R)-1-Hydroxypropan-2-yl)-13-methyleicosa-5,8,11,14-tetraenamide
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(3a). To a solution of 37a (25 mg, 0.04 mmol) in dry THF (0.8 mL) at 0 ºC under an argon atmosphere,
was added TBAF (0.05 mL, 1M solution in THF) dropwise. The reaction mixture was stirred for 10
min at 0 ºC and for 1.5 hours at room temperature, and then it was quenched with saturated aqueous
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NH Cl at 0 ºC and extracted with EtOAc. The combined organic extracts were washed with brine, dried
4
over MgSO and the solvent was evaporated under reduced pressure at 35-37 ºC. The residue was
4
purified by flash column chromatography on silica gel (40% EtOAc/hexane) to give 3a (13 mg, 87 %
-1
yield) as a colorless oil. IR (neat): 3300, 3019, 2926, 2861, 1644 (C=O), 1540, 1273, 1262, 1069 cm ;
1
H NMR (600 MHz, CDCl ) δ 0.89 (t, J = 6.7 Hz, 3H, 20-H), 1.02 (d, J = 6.6 Hz, 3H, =CH-CH(CH )-
3
3
), 1.17 (d, J = 6.6 Hz, 3H, >N-CH(CH )-), 1.24 – 1.32 (m, 4H, 18-H, 19-H), 1.35 (sextet, J = 7.2 Hz,
3
2
H, 17-H), 1.72 (quintet, J = 7.2 Hz, 2H, 3-H), 2.06 (nonet, J = 6.2 Hz, 2H, 16-H), 2.12 (dt, J = 7.2 Hz,
J = 7.2 Hz, 2H, 4-H), 2.20 (t, J = 7.8 Hz, 2H, 2-H), 2.76 – 2.84 (ddd and t, overlapping, 3H, 10-H and
7
3
-H, especially 2.81, t, J = 6.0 Hz, 2H, 7-H), 2.87 (ddd, J = 15.0 Hz, J = 7.5 Hz, J = 6.2 Hz, 1H, 10-H),
.46 (sextet, J = 7.5 Hz, 1H, 13-H), 3.53 (dd, J = 10.8 Hz, J = 6.0 Hz, 1H, -CH -O-), 3.67 (dd, J = 10.8
2
Hz, J = 3.6 Hz, 1H, -CH -O-), 3.68 (br s, 1H, -OH), 4.08 (m, 1H, >N-CH(CH )-), 5.20 – 5.30 (m, 4H,
2
3
1
3
1
1-H, 12-H, 14-H, 15-H), 5.32 – 5.44 (m, 4H, 5-H, 6-H, 8-H, 9-H), 5.60 (br s, 1H, >NH). C NMR (75
MHz, CDCl ) δ 173.7 (>C=O), 135.1 (C-12), 134.1 (C-14), 129.0 (C-8), 128.8 (C-5), 128.4 (C-6),
3
1
28.2 (C-15), 128.1 (C-9), 125.6 (C-11), 67.4 (-CH -OH), 47.8 (-NH-CH<), 36.1 (C-2), 31.6 (C-18),
2
30.5 (C-13), 29.5 (C-17), 27.5 (C-16), 26.6 (C-4), 25.8 (C-10), 25.6 (C-7), 25.5 (C-3), 22.6 (C-19), 22.0
(C-13-CH ), 17.1 (-NH-CH(CH )-), 14.1 (C-20); Mass spectrum (EI) m/z (relative intensity) 376
3 3
+
+
(
M+H˥ , 1), 375 (M , 2), 357 (5), 342 (7), 232 (8), 192 (12), 152 (15), 139 (22), 112 (39), 99 (100);
+
Exact mass (EI) calculated for C H NO (M+H˥ ), 376.3216; found, 376.3213; Mass spectrum (ESI)
2
4
42
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+
m/z (relative intensity) 376 (M+H˥ , 100), 339 (15), 301 (11), 269 (13); Exact mass (ESI) calculated
+
for C H NO (M+H˥ ), 376.3216; found, 376.3210. LC/MS analysis (Waters MicroMass ZQ system)
2
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42
2
showed purity 98% and retention time 5.5 min for the title compound.
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(
13S,5Z,8Z,11Z,14Z)-N-((R)-1-Hydroxypropan-2-yl)-13-methyl-18-phenoxyoctadeca-5,8,11,14-
tetraenamide (3b). The synthesis was carried out as described for 3a, using 37b (22 mg, 0.036 mmol)
and TBAF (0.045 mL, 1M solution in THF) in dry THF (0.7 mL). The reaction was completed in 1.5
hours and the crude oil obtained after work up was purified by flash column chromatography on silica
1
gel (57:40:3 ethyl acetate:hexane:MeOH) to give 3b (12 mg, 85 % yield) as a colorless oil. H NMR
(600 MHz, CDCl ) δ 0.99 (d, J = 7.2 Hz, 3H, =CH-CH(CH )-), 1.15 (d, J = 6.6 Hz, 3H, >N-CH(CH )-),
3
3
3
1
.71 (quintet, J = 7.2 Hz, 2H, 3-H), 1.85 (m, 2H, 17-H), 2.10 (dt, J = 6.6 Hz, J = 6.6 Hz, 2H, 4-H), 2.17
(
t, J = 7.2 Hz, 2H, 2-H), 2.20 – 2.26 (m, 1H, 16-H), 2.26 – 2.34 (m, 1H, 16-H), 2.75 – 2.85 (ddd and t,
overlapping, 3H, 10-H and 7-H, especially 2.80, t, J = 6.0 Hz, 2H, 7-H), 2.87 (ddd, J = 15.6 Hz, J = 7.5
Hz, J = 6.5 Hz, 1H, 10-H), 3.49 (ddq as sextet, J = 7.2 Hz, 1H, 13-H), 3.51 (dd, J = 10.8 Hz, J = 6.3
Hz, 1H, -CH -OH), 3.65 (dd, J = 10.8 Hz, J = 3.3 Hz, 1H, -CH -OH), 3.96 (t, J = 6.6 Hz, 2H, 18-H),
2
2
4
.06 (m, 1H, >N-CH(CH )-), 5.20 – 5.42 (m, 8H, 5-H, 6-H, 8-H, 9-H, 11-H, 12-H, 14-H, 15-H), 5.57
3
(
br s, 1H, >NH), 6.89 (d, J = 7.4 Hz, 2H, 2-H, 6-H, -OPh), 6.93 (t, J = 7.4 Hz, 1H, 4-H, -OPh), 7.27 (t,
13
J = 7.4 Hz, 2H, 3-H, 5-H, -OPh). C NMR (75 MHz, CDCl ) δ 173.7 (>C=O), 159.0 (C-1ʹ, Ar), 135.3,
3
1
34.8, 129.4 (C-3ʹ, C-5ʹ, Ar), 129.1, 128.8, 128.3, 128.1, 126.8, 125.8, 120.5 (C-4ʹ, Ar), 114.5 (C-2ʹ, C-
6ʹ, Ar), 67.5 (-CH O-), 67.0 (-CH O-), 47.8 (-NH-CH<), 36.0 (C-2), 30.5, 29.2, 26.6, 25.8, 25.6, 25.4,
2
2
+
4.0, 21.9, 17.0 (-NH-CH(CH )-); Mass spectrum (EI) m/z (relative intensity) 439 (M , 4), 421 (15),
2
3
4
06 (8), 328 (9), 300 (8), 232 (13), 192 (14), 152 (19), 139 (35), 112 (37), 99 (100); Exact mass (EI)
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calculated for C H NO , 439.3086; found, 439.3083. LC/MS analysis (Waters MicroMass ZQ
2
8
41
3
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system) showed purity 97% and retention time 5.2 min for the title compound.
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(13R,5Z,8Z,11Z,14Z)-N-((R)-1-Hydroxypropan-2-yl)-13-methyleicosa-5,8,11,14-tetraenamide
(3c). The synthesis was carried out as described for 3a, using 37c (14 mg, 0.023 mmol) and TBAF
(0.03 mL, 0.03 mmol, 1 M solution in THF) in dry THF (0.5 mL). The reaction was completed in 1.5
hours and the crude product obtained after work up was purified by flash column chromatography on
silica gel (57:40:3 ethyl acetate:hexane:MeOH) to give 3c (7 mg, 80 % yield) as a colorless oil. IR
-
1
1
neat): 3304, 3013, 2927, 2857, 1653 (C=O), 1543, 1275, 1261, 1066 cm ; H NMR (600 MHz,
(
CDCl ) δ 0.89 (t, J = 6.7 Hz, 3H, 20-H), 1.02 (d, J = 6.6 Hz, 3H, =CH-CH(CH )-), 1.17 (d, J = 6.6 Hz,
3
3
3
H, >N-CH(CH )-), 1.24 – 1.32 (m, 4H, 18-H, 19-H), 1.35 (sextet, J = 7.2 Hz, 2H, 17-H), 1.72 (quintet,
3
J = 7.2 Hz, 2H, 3-H), 2.06 (nonet, J = 6.2 Hz, 2H, 16-H), 2.12 (dt, J = 7.2 Hz, J = 7.2 Hz, 2H, 4-H),
2.20 (t, J = 7.8 Hz, 2H, 2-H), 2.79 – 2.85 (ddd and t, overlapping, 3H, 10-H and 7-H, especially 2.81, t,
J = 6.0 Hz, 2H, 7-H), 2.87 (ddd, J = 15.0 Hz, J = 7.5 Hz, J = 6.2 Hz, 1H, 10-H), 3.46 (sextet, J = 7.5
Hz, 1H, 13-H), 3.53 (dd, J = 10.8 Hz, J = 6.0 Hz, 1H, -CH -O-), 3.67 (dd, J = 10.8 Hz, J = 3.6 Hz, 1H,
2
-CH -O-), 3.68 (br s, 1H, -OH), 4.08 (m, 1H, >N-CH(CH )-), 5.20 – 5.31 (m, 4H, 11-H, 12-H, 14-H,
2 3
1
3
1
1
5-H), 5.32 – 5.44 (m, 4H, 5-H, 6-H, 8-H, 9-H), 5.55 (br s, 1H, >NH). C NMR (75 MHz, CDCl ) δ
3
73.7 (>C=O), 135.1 (C-12), 134.1 (C-14), 129.0 (C-8), 128.8 (C-5), 128.4 (C-6), 128.2 (C-15), 128.1
(C-9), 125.6 (C-11), 67.4 (-CH -OH), 47.8 (-NH-CH<), 36.1 (C-2), 31.6 (C-18), 30.5 (C-13), 29.4 (C-
2
17), 27.5 (C-16), 26.6 (C-4), 25.8 (C-10), 25.6 (C-7), 25.5 (C-3), 22.6 (C-19), 22.0 (C-13-CH ), 17.1 (-
3
+
NH-CH(CH )-), 14.1 (C-20). MS (EI): m/z (%): 375 (M , 1), 374 (2), 312 (11), 284 (17), 257 (23), 244
3
(30), 171 (41), 57 (100); Exact mass (EI) calculated for C H NO , 375.3137; found, 375.3128; MS
24 41 2
+
(ESI): m/z (%): 376 (M+H˥ , 52), 339 (84), 269 (100); Exact mass (ESI) calculated for C H NO
2
4
42
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+
(
M+H˥ ), 376.3216; found, 376.3203. LC/MS analysis (Waters MicroMass ZQ system) showed purity
9
8% and retention time 5.5 min for the title compound.
0
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(
7R,5Z,8Z,11Z,14Z)-N-((R)-1-Hydroxypropan-2-yl)-7-methyleicosa-5,8,11,14-tetraenamide (3d).
The synthesis was carried out as described for 3a, using 37d (20 mg, 0.032 mmol) and TBAF (0.03
mL, 0.03 mmol, 1 M solution in THF) in dry THF (0.5 mL). The reaction was completed in 1.5 hours
and the crude product obtained after work up was purified by flash column chromatography on silica
gel (57:40:3 ethyl acetate:hexane:MeOH) to give 3d (10 mg, 85 % yield) as a colorless oil. IR (neat):
-1 1
3
302, 3011, 2927, 2857, 1647 (C=O), 1542, 1270, 1261, 1061 cm ; H NMR (500 MHz, CDCl ) δ 0.89
3
(t, J = 6.9 Hz, 3H, 20-H), 1.02 (d, J = 6.7 Hz, 3H, =CH-CH(CH )-), 1.17 (d, J = 6.7 Hz, 3H, >CH-
3
CH ), 1.23 – 1.42 (m, 6H, 17-H, 18-H, 19-H), 1.67 – 1.78 (quintet, J = 7.0 Hz, 2H, 3-H), 2.06 (dt, J =
3
7
.0 Hz, J = 7.0 Hz, 2H, 16-H), 2.09 – 2.17 (m, 2H, 4-H), 2.19 (t, J = 7.5 Hz, 2H, 2-H), 2.75 – 2.91 (m,
4H, 10-H, 13-H), 3.44 (sextet, J = 7.0 Hz, 1H, 7-H), 3.53 (m as qtd, J = 10.8 Hz, J = 3.7 Hz, 1H, -
CH OH), 3.66 (m as br d, J = 10.8 Hz, 1H, -CH OH), 4.07 (m, 1H, >CH(CH )), 5.20 – 5.45 (m, 8H, 5-
2
2
3
13
H, 6-H, 8-H, 9-H, 11-H, 12-H, 14-H, 15-H), 5.56 (br d, J = 5.3 Hz, 1H, >NH). C NMR (100 MHz,
CDCl ) δ 173.6 (C=O), 135.3 (C=C), 134.7 (C=C), 130.5 (C=C), 128.6 (C=C), 128.0 (C=C), 127.5
3
(
C=C), 126.8 (C=C), 125.9 (C=C), 67.5 (CH OH), 47.9 (-NHCH<), 36.1, 31.5, 30.5, 29.3, 27.2, 26.9,
2
+
5.8, 25.6, 25.5, 22.5, 22.0, 17.0, 14.0. Mass spectrum (ESI) m/z (relative intensity) 377 (M + 2H, 25),
2
+
76 (M + H, 100), 301 (8). Exact mass (ESI) calculated for C H NO (M + H), 376.3216; found,
+
3
2
4
42
2
376.3208. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98% and retention time 5.5
min for the title compound.
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(
7S,5Z,8Z,11Z,14Z)-N-((R)-1-Hydroxypropan-2-yl)-7-methyleicosa-5,8,11,14-tetraenamide (3e).
The synthesis was carried out as described for 3a, using 37e (20 mg, 0.032 mmol) and TBAF (0.03 mL,
0.03 mmol, 1 M solution in THF) in dry THF (0.5 mL). The reaction was completed in 1.5 hours and
the crude product obtained after work up was purified by flash column chromatography on silica gel
6
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(57:40:3 ethyl acetate:hexane:MeOH) to give 3e (10 mg, 81 % yield) as a colorless oil. IR (neat): 3304,
-
1 1
3
012, 2927, 2857, 1650 (C=O), 1543, 1278, 1264, 1059 cm ; H NMR (500 MHz, CDCl ) δ 0.89 (t, J
3
=
6.9 Hz, 3H, 20-H), 1.02 (d, J = 6.7 Hz, 3H, =CH-CH(CH )-), 1.17 (d, J = 6.7 Hz, 3H, >CH-CH ),
3 3
1
.23 – 1.42 (m, 6H, 17-H, 18-H, 19-H), 1.67 – 1.78 (quintet, J = 7.0 Hz, 2H, 3-H), 2.06 (dt, J = 7.0 Hz,
J = 7.0 Hz, 2H, 16-H), 2.09 – 2.17 (m, 2H, 4-H), 2.19 (t, J = 7.5 Hz, 2H, 2-H), 2.75 – 2.91 (m, 4H, 10-
H, 13-H), 3.44 (sextet, J = 7.0 Hz, 1H, 7-H), 3.53 (m as qtd, J = 10.8 Hz, J = 3.7 Hz, 1H, -CH OH),
2
3
.66 (m as br d, J = 10.8 Hz, 1H, -CH OH), 4.07 (m, 1H, >CH(CH )), 5.20 – 5.45 (m, 8H, 5-H, 6-H, 8-
2 3
1
3
H, 9-H, 11-H, 12-H, 14-H, 15-H), 5.56 (br d, J = 5.3 Hz, 1H, >NH). C NMR (100 MHz, CDCl ) δ
3
173.7 (C=O), 135.4 (C=C), 134.7 (C=C), 130.6 (C=C), 128.6 (C=C), 128.0 (C=C), 127.5 (C=C), 126.8
(C=C), 125.9 (C=C), 67.5 (CH OH), 47.9 (NHCH), 36.2, 31.5, 30.5, 29.3, 27.2, 26.9, 25.8, 25.6, 25.6,
2
+
2.5, 22.0, 17.0, 14.0. Mass spectrum (ESI) m/z (relative intensity) 398 (M + Na, 8), 376 (M + H,
+
2
+
00), 301 (8). Exact mass (ESI) calculated for C H NO (M + H), 376.3216; found, 376.3206.
1
24 42
2
LC/MS analysis (Waters MicroMass ZQ system) showed purity 98% and retention time 5.5 min for the
title compound.
(13S,5Z,8Z,11Z,14Z)-N-Cyclopropyl-13-methyleicosa-5,8,11,14-tetraenamide (4a). A solution of
(13S,5Z,8Z,11Z,14Z)-13-methyleicosa-5,8,11,14-tetraenoic acid (34a, 12 mg, 0.04 mmol), and dried
carbonyldiimidazole (18 mg, 0.113 mmol) in dry THF (0.63 mL) at room temperature under an argon
atmosphere, was stirred for 2 hours and then a solution of cyclopropyl amine (9 mg, 0.151 mmol) in
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THF (0.2 mL) was added. The reaction mixture was stirred for 1 hour and then diluted with water and
ethyl acetate. The organic phase was separated, and the aqueous phase extracted with AcOEt. The
combined organic layer was washed with brine, dried over MgSO and concentrated under reduced
4
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0
pressure. Purification by flash column chromatography on silica gel (25 % acetone in hexane) gave 12
1
mg (91 % yield) of 4a as a colorless oil. H NMR (500 MHz, CDCl ) δ 0.47 (m, 2H of the cyclopropyl
3
ring), 0.76 (m, 2H of the cyclopropyl ring), 0.89 (t, J = 6.9 Hz, 3H, 20-H), 1.02 (d, J = 6.6 Hz, 3H,
>CH-CH ), 1.24 – 1.33 (m, 4H, 18-H, 19-H), 1.35 (sextet, J = 7.2 Hz, 2H, 17-H), 1.71 (quintet, J = 7.2
3
Hz, 2H, 3-H), 2.02 – 2.09 (m, 2H, 16-H), 2.10 (dt, J = 7.2 Hz, J = 7.2 Hz, 2H, 4-H), 2.12 (t, J = 7.8 Hz,
H, 2-H), 2.70 (octet, J = 3.6 Hz, 1H, >N-CH<), 2.75 – 2.85 (ddd and t overlapping, 3H, 10-H and 7-H,
especially 2.80, t, J = 6.6 Hz, 2H, 7-H), 2.87 (ddd, J = 15.0 Hz, J = 7.3 Hz, J = 6.6 Hz, 1H, 10-H), 3.46
2
(sextet, J = 7.5 Hz, 1H, 13-H), 5.20 – 5.30 (m, 4H, 11-H, 12-H, 14-H, 15-H), 5.31 – 5.43 (m, 4H, 5-H,
1
3
6
-H, 8-H, 9-H), 5.51 (br s, 1H, >NH). C NMR (100 MHz, CDCl ) δ 174.1 (C=O), 135.1 (C=C), 134.1
3
(C=C), 129.1 (C=C), 128.7 (C=C), 128.4 (C=C), 128.2 (C=C), 128.1 (C=C), 125.6 (C=C), 35.9 (-NH-
CH<), 31.5, 30.5, 29.4, 27.5, 26.6, 25.8, 25.6, 25.4, 22.5, 22.0, 14.0, 6.6. Mass spectrum (EI) m/z
+
relative intensity) 357 (M , 5), 300 (4), 286 (5), 232 (10), 117 (7), 175 (11), 147 (10), 133 (17), 119
(
(17), 107 (22), 99 (39), 93 (54), 57 (100); Exact mass (EI) calculated for C H NO, 357.3032; found,
24 39
3
57.3025. LC/MS analysis (Waters MicroMass ZQ system) showed purity 97% and retention time 6.1
min for the title compound.
(13S,5Z,8Z,11Z,14Z)-N-Cyclobutyl-13-methyleicosa-5,8,11,14-tetraenamide (4b). The synthesis
was carried out as described for 4a, using 34a (24 mg, 0.08 mmol), fresh carbonyldiimidazole (37 mg,
0
.226 mmol) and cyclobutyl amine (22 mg, 0.302 mmol) in dry THF (0.4 mL). The crude oil obtained
after work up was purified by flash column chromatography on silica gel (16% acetone in hexane) and
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gave 4b (26 mg, 92 % yield) as a colorless oil. H NMR (600 MHz, CDCl ) δ 0.89 (t, J = 6.9 Hz, 3H,
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
0-H), 1.02 (d, J = 6.6 Hz, 3H, >CH-CH ), 1.25 – 1.33 (m, 4H, 18-H, 19-H), 1.36 (sextet, J = 7.2 Hz,
3
2H, 17-H), 1.65 – 1.74 (m and quintet overlapping, 4H, cyclobutane ring and 3-H, especially 1.71,
0
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quintet, J = 7.2 Hz, 2H, 3-H), 1.78 – 1.86 (m, 2H, cyclobutane ring), 2.03 – 2.09 (m, 2H, 16-H), 2.10
(
dt, J = 7.2 Hz, J = 7.2 Hz, 2H, 4-H), 2.13 (t, J = 7.2 Hz, 2H, 2-H), 2.30 – 2.38 (m, 2H, cyclobutane
ring), 2.75 – 2.85 (ddd and t overlapping, 3H, 10-H and 7-H, especially 2.80, t, J = 6.2 Hz, 2H, 7-H),
.87 (ddd, J = 15.0 Hz, J = 7.4 Hz, J = 6.7 Hz, 1H, 10-H), 3.46 (sextet, J = 7.7 Hz, 1H, 13-H), 4.10
sextet, J = 8.4 Hz, 1H, >N-CH<), 5.20 – 5.30 (m, 4H, 11-H, 12-H, 14-H, 15-H), 5.31 – 5.43 (m, 4H, 5-
2
(
1
3
H, 6-H, 8-H, 9-H), 5.52 (br s, 1H, >NH). C NMR (75 MHz, CDCl ) δ 171.8 (>C=O), 135.1 (C-12),
3
134.1 (C-14), 129.1 (C-8), 128.7 (C-5), 128.4 (C-6), 128.2 (C-15), 128.1 (C-9), 125.6 (C-11), 44.6 (-
NH-CH<), 36.0 (C-2), 31.6 (C-18), 31.4 (cyclobutane ring), 30.5 (C-13), 29.4 (C-17), 27.5 (C-16), 26.6
(C-4), 25.8 (C-10), 25.6 (C-7), 25.4 (C-3), 22.6 (C-19), 22.0 (C-13-CH ), 15.0 (cyclobutane ring), 14.1
3
+
(C-20); Mass spectrum (ESI) m/z (relative intensity) 372 (M+H˥ , 100), 301 (5); Exact mass (ESI)
+
calculated for C H NO (M+H˥ ), 372.3266; found, 372.3264. LC/MS analysis (Waters MicroMass
2
5
42
ZQ system) showed purity 98% and retention time 6.1 min for the title compound.
(
13S,5Z,8Z,11Z,14Z)-N-Cyclopentyl-13-methyleicosa-5,8,11,14-tetraenamide (4c). The synthesis
was carried out as described for 4a, using 34a (24 mg, 0.08 mmol), fresh carbonyldiimidazole (37 mg,
.226 mmol) and cyclopentyl amine (26 mg, 0.302 mmol) in dry THF (0.4 mL). The crude oil obtained
0
after work up was purified by flash column chromatography on silica gel (20 % acetone in hexane) and
1
gave 4c (25 mg, 85 % yield) as a colorless oil. H NMR (600 MHz, CDCl ) δ 0.88 (t, J = 7.2 Hz, 3H,
3
2
0-H), 1.02 (d, J = 6.6 Hz, 3H, >CH-CH ), 1.24 – 1.39 (m, 8H, cyclopentane ring, 17-H, 18-H, 19-H),
3
1.56 – 1.62 (m, 2H, cyclopentane ring), 1.62 – 1.69 (m, 2H, cyclopentane ring), 1.70 (quintet, J = 7.5
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Hz, 2H, 3-H), 1.99 (sextet, J = 6.3 Hz, 2H, cyclopentane ring), 2.03 – 2.09 (m, 2H, 16-H), 2.11 (dt, J =
.3 Hz, J = 7.3 Hz, 2H, 4-H), 2.14 (t, J = 7.8 Hz, 2H, 2-H), 2.75 – 2.85 (ddd and t overlapping, 3H, 10-
H and 7-H, especially 2.80, t, J = 6.0 Hz, 2H, 7-H), 2.87 (ddd, J = 15.0 Hz, J = 7.4 Hz, J = 6.7 Hz, 1H,
7
0
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6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
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4
5
6
7
8
9
0
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1
0-H), 3.46 (sextet, J = 7.8 Hz, 1H, 13-H), 4.21 (sextet, J = 7.2 Hz, 1H, >N-CH<), 5.20 – 5.30 (m, 4H,
13
1-H, 12-H, 14-H, 15-H), 5.31 – 5.43 (m, 5H, >NH, 5-H, 6-H, 8-H, 9-H). C NMR (75 MHz, CDCl )
1
3
δ 172.32 (>C=O), 135.1 (C-12), 134.1 (C-14), 129.2 (C-8), 128.7 (C-5), 128.4 (C-6), 128.2 (C-15),
1
1
28.1 (C-9), 125.6 (C-11), 51.1 (-NH-CH<), 36.2 (C-2), 33.2 (cyclopentane ring), 31.6 (C-18), 30.5 (C-
3), 29.4 (C-17), 27.5 (C-16), 26.6 (C-4), 25.8 (C-10), 25.6 (C-7 or C-3), 25.6 (C-3 or C-7), 23.7
(cyclopentane ring), 22.6 (C-19), 22.0 (C-13-CH ), 14.1 (C-20); Mass spectrum (ESI) m/z (relative
3
+
+
intensity) 386 (M+H˥ , 100), 184 (9); Exact mass (ESI) calculated for C H NO (M+H˥ ), 386.3423;
2
6
44
found, 386.3414. LC/MS analysis (Waters MicroMass ZQ system) showed purity 98% and retention
time 6.1 min for the title compound.
(
13S,5Z,8Z,11Z,14Z)-13-Methyl-N-(prop-2-yn-1-yl)eicosa-5,8,11,14-tetraenamide
synthesis was carried out as described for 4a, using 34a (12 mg, 0.04 mmol), fresh carbonyldiimidazole
18 mg, 0.113 mmol) and propargyl amine (8.3 mg, 0.151 mmol) in dry THF (0.2 mL). The crude oil
(4d).
The
(
obtained after work up was purified by flash column chromatography on silica gel (25 % acetone in
1
hexane) and gave 4d (12 mg, 88 % yield) as a colorless oil. H NMR (600 MHz, CDCl ) δ 0.89 (t, J =
3
6
.6 Hz, 3H, 20-H), 1.02 (d, J = 6.6 Hz, 3H, >CH-CH ), 1.24 – 1.33 (m, 4H, 18-H, 19-H), 1.37 (sextet, J
3
= 7.2 Hz, 2H, 17-H), 1.73 (quintet, J = 7.8 Hz, 2H, 3-H), 2.06 (nonet, J = 7.2 Hz, 2H, 16-H), 2.12 (dt, J
7.2 Hz, J = 7.2 Hz, 2H, 4-H), 2.20 (t, J = 7.8 Hz, 2H, 2-H), 2.23 (t, J = 2.4 Hz, 1H, -C≡CH), 2.75 –
.84 (ddd and t overlapping, 3H, 10-H and 7-H, especially 2.81, t, J = 6.0 Hz, 2H, 7-H), 2.87 (ddd, J =
=
2
15.0 Hz, J = 7.5 Hz, J = 6.2 Hz, 1H, 10-H), 3.46 (sextet, J = 7.8 Hz, 1H, 13-H), 4.05 (dd, J = 4.8 Hz, J
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=
2.4 Hz, 2H, -CH -C≡C-), 5.20 – 5.30 (m, 4H, 11-H, 12-H, 14-H, 15-H), 5.32 – 5.44 (m, 4H, 5-H, 6-
2
1
3
H, 8-H, 9-H), 5.55 (br s, 1H, >NH). C NMR (75 MHz, CDCl ) δ 172.3 (>C=O), 135.1 (C-12), 134.1
3
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
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3
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5
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5
6
(C-14), 128.9 (C-8), 128.9 (C-5), 128.4 (C-6), 128.2 (C-15), 128.1 (C-9), 125.6 (C-11), 79.5 (-C≡CH),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
1.6 (-C≡CH), 35.7 (C-2), 31.5 (C-18), 30.5 (C-13), 29.4 (C-17), 29.1 (-NH-CH -), 27.5 (C-16), 26.5
2
(
(
(
C-4), 25.8 (C-10), 25.6 (C-7), 25.2 (C-3), 22.6 (C-19), 22.0 (C-13-CH ), 14.0 (C-20); Mass spectrum
3
+
ESI) m/z (relative intensity) 356 (M+H˥ , 100), 301 (10); Exact mass (ESI) calculated for C H NO
24 38
+
M+H˥ ), 356.2953; found, 356.2953. LC/MS analysis (Waters MicroMass ZQ system) showed purity
9
8% and retention time 6.0 min for the title compound.
(13S,5Z,8Z,11Z,14Z)-N-(2-Chloroethyl)-13-methyleicosa-5,8,11,14-tetraenamide
synthesis was carried out as described for 4a, using 34a (25 mg, 0.08 mmol), fresh carbonyldiimidazole
38 mg, 0.236 mmol) and a mixture of chloroethyl amine hydrochloride 37 mg, 0.314 mmol) and dry
(4e).
The
(
pyridine (0.3 mL) in THF (0.4 mL). The crude oil obtained after work up was purified by flash column
chromatography on silica gel (20 % acetone in hexane) and gave 4e (27 mg, 89 % yield) as a colorless
1
oil. H NMR (600 MHz, CDCl ) δ 0.89 (t, J = 6.7 Hz, 3H, 20-H), 1.02 (d, J = 6.6 Hz, 3H, >CH-CH ),
3
3
1
.24 – 1.33 (m, 4H, 18-H, 19-H), 1.36 (sextet, J = 7.2 Hz, 2H, 17-H), 1.73 (quintet, J = 7.8 Hz, 2H, 3-
H), 2.06 (nonet, J = 6.6 Hz, 2H, 16-H), 2.12 (dt, J = 7.2 Hz, J = 7.2 Hz, 2H, 4-H), 2.22 (t, J = 7.8 Hz, 2-
H), 2.76 –2.84 (ddd and t overlapping, 3H, 10-H and 7-H, especially 2.81, t, J = 6.0 Hz, 2H, 7-H), 2.87
(ddd, J = 15.0 Hz, J = 7.5 Hz, J = 6.2 Hz, 1H, 10-H), 3.46 (sextet, J = 7.4 Hz, 1H, 13-H), 3.58 – 3.65
(m and t overlapping, 4H, >N-CH -, -CH -Cl, especially 3.62, t, J = 4.8 Hz, 2H, -CH -Cl), 5.20 – 5.31
2
2
2
13
m, 4H, 11-H, 12-H, 14-H, 15-H), 5.33 – 5.44 (m, 4H, 5-H, 6-H, 8-H, 9-H), 5.84 (br s, 1H, >NH). C
(
NMR (75 MHz, CDCl ) δ 173.0 (>C=O), 135.1 (C-12), 134.1 (C-14), 129.0 (C-8), 128.9 (C-5), 128.4
3
(C-6), 128.2 (C-15), 128.1 (C-9), 125.6 (C-11), 44.2 (-CH Cl), 41.1 (-NH-CH -), 35.9 (C-2), 31.6 (C-
2
2
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