Multicomponent Approach to Libraries of Substituted Dihydroorotic Acid Amides

Article abstract of DOI:10.1021/acscombsci.9b00144

A process featuring a sequential multicomponent reaction followed by a regioselective postcyclization strategy was implemented for the facile synthesis of N,N′-disubstituted dihydroorotic acid amides under mild conditions. We obtained, for the first time, a library of 29 derivatives, encompassing 19 N^α-substituted-N⁴-dihydroorotyl-4-aminophenylalanine derivatives, a key residue of gonadotropin-releasing hormone antagonist Degarelix. The corresponding products were prepared from easily accessible starting materials in good to excellent yields with broad substrate scope.

Full text of DOI:10.1021/acscombsci.9b00144

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Article
Multicomponent Approach to Libraries of
Substituted Dihydroorotic Acid Amides
Maria Cristina Bellucci, Alessandro Sacchetti, and Alessandro Volonterio
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.9b00144 • Publication Date (Web): 27 Aug 2019
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Multicomponent Approach to Libraries of Substituted
Dihydroorotic Acid Amides
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Maria Cristina Bellucci,§ Alessandro Sacchetti,† Alessandro Volonterio†,*
Department of Chemistry, Material and Chemical Engineer “Giulio Natta”, Politecnico di Milano,
via Mancinelli 7, 20131 Milano, Italy; Department of Food, Environmental and Nutritional
Sciences, Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy.
alessandro.volonterio@polimi.it
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
§ Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano
† Department of Chemistry, Material and Chemical Engineer “Giulio Natta”, Politecnico di Milano.
Abstract
A process featuring a sequential multicomponent reaction followed by a regioselective post-
cyclization strategy was implemented for the facile synthesis of N,N’-disubstituted dihydroorotic acid
amides under mild conditions. We obtained, for the first time, a library of 29 derivatives,
encompassing 19 Nα-substituted-N4-dihydroorotyl-4-aminophenylalanine derivatives, a key residue
of gonadotropin-releasing hormone antagonist Degarelix. The corresponding products were prepared
from easily accessible starting materials in good to excellent yields with broad substrate scope.
KEYWORDS Combinatorial Chemistry; Multicomponent Reactions; Domino Process;
Carbodiimide; Dihydroorotic acid.
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INTRODUCTION
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Dihydroorotic acid (2,6-dioxo-4-hexahydro-pyrimidinecarboxylic acid, DHO acid 1, Figure 1)
derivatives are chemically and biologically very interesting compounds since DHO acid is a key
component of pyrimidine nucleotide biosynthesis.1 DHO acid is formed by cyclization of N-
carbamoyl-aspartic acid precursor triggered by zinc metalloenzyme dihydroorotase (DHOase) and
converted to orotic acid by an oxidation catalyzed by the dihydroorotate dehydrogenase (DHODH)
enzyme.2 DHO acid derivatives able to target and inhibit DHOase or DHODH are potential
anticancer and antimalarial drugs.3 Since the biological activity of heterocycles in general often do
not arise only from the heterocycle itself, but from the substituents attached to it, the development of
a practical and straightforward way to synthesize libraries of substituted DHO acid derivatives is of
great interest.
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Moreover, DHO acid is found in marketed drugs, such as Degarelix 2, a decapeptide where N4-
(dihydroorotyl)-4-aminophenylalanine (4-Aph(Hor)) amino acid is incorporated in position 5.
(Figure 1).
O
NH
HN
O
O
NH
N
O
NH
HN
O
O
O
O
O
O
H
H
H
H
O
NH2
O
N
N
N
N
N
N
N
N
N
N
H
OH
DHO acid
H
H
H
H
O
O
O
O
O
OH
1
NH
NH2
Cl
NH
2
O
Degarelix
Figure 1. Structures of DHO acid 1 and Degarelix 2.
The introduction of a DHO acid residue, along with a urea moiety in position 5 and 6, respectively,
of known peptides acting as gonadotropin-releasing hormone (GnRH) antagonists, led to the
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discovery of analogues with improved hydrophilicity, lesser propensity to form gels, and a dramatic
increase in duration of action when submitted subcutaneously.4 However, so far there is not a
mechanistic insight to explain these unique features of Degarelix and new studies are still ongoing.5
Since the introduction of hydrophobic side chains, as well as new hydrogen bonding sites, may
modulate the activity/solubility/bioavailability, the incorporation of N-substituted DHO acid in
Degarelix, or more general in GnRH antagonist of similar structure, could be an important goal in
order to shed light on the relationship between the peptide’s structure and the morphology of the self-
assembled nanostructures and/or to find biologically and pharmaceutically more active peptides.
Thus, also for this purpose, the development of a process that would make accessible libraries of
substituted 4-Aph(Hor) derivatives is of great interest.
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However, an extensive literature survey revealed that there are few reports describing the synthesis
of N-substituted DHO acid. To date, the most utilized strategies rely in a step-wise
ethoxycarbonylation of N-substituted asparagine followed by cyclization under strongly basic
conditions6 or by alkylation of DHO esters using strong bases such as NaH or potassium tert-
butoxide in dry solvents and highly reactive methyl or benzyl halides (or more reactive
trifluoromethansulphonates), followed by ester saponification.7
The most explored tool in drug discovery programs, both in industry and in academia, for the
combinatorial synthesis of new entities in a programmed and efficient manner, and in particular for
heterocycles, is the application of multicomponent reactions (MCRs), which allow the simultaneous
formation of multiple bonds starting from three or more reactants in one-step.8 However, the use of
MCR strategy suffers from a key issue, i.e. a limited scaffold diversity due to the difficulty to
discover a new MC process.9 In particular, to the best of our knowledge, there are no reports in the
literature describing the combinatorial synthesis of DHO acid derivatives either by MCRs or by
multi-step synthetic pathways.
During the last years, we have developed a new sequential MCR that allowed us to prepare
libraries of hydantoin derivatives and urea-peptide conjugates incorporating a hexafluorovaline or
aspartic (Asp) ester moieties starting from easily accessible reagents under very mild conditions.10 In
order to exploit known MCRs to increase scaffold diversity and complexity, along with the single
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reactant replacement (SRR)11 and condition-based divergence (CBD)12 strategies, a particularly
attractive and synthetically productive manipulation of MCRs is the post-cyclization strategy which
triggers the formation of (poly)cyclic compounds, mainly (poly)heterocycles.13 In this Research
Article, we present for the first time the combinatorial synthesis of amides and peptides
incorporating substituted DHO or 4Aph(Hor) moieties, which arises by fast and regioselective, one-
pot, post-cyclization of N-urea-aspartate derivatives obtained by our MCR.
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RESULTS AND DISCUSSION
We recently demonstrated that carbodiimides 7, generated in situ from azides 3 and
iso(thio)cyanates 4 through Staudinger/aza-Wittig process, react straightforwardly in the presence of
fumaric acid monoesters 5 and amines or aminoacids 6 leading to the formation of, respectively,
urea-amide or -peptide conjugates incorporating an aspartic unit 8 (Scheme 1).10 In a previous work
concerning the application of our MC process to the synthesis of neomycin-sugar conjugates, when
the conjugates were treated with a solution of methylamine, along with sugar de-acetylation, we
obtained the immediate formation of a DHO heterocycle probably through the nucleophilic attack of
the urea NH to the ester function of the Asp unit.14 This result was quite unexpected since cyclization
of ureido-esters to pyrimidine-2,4-diones was known to occur with strong bases (such as t-BuOK or
NaH) or weaker inorganic bases (CsCO3) in very polar solvent, DMF, and long reaction time
(typically over-night). We decided to study in depth this process by treating urea-Asp amide
conjugates 8a-g in the same reaction conditions used for the MC process, i.e. by dissolving the
adducts 8 in CH3CN and adding a 10% in volume of a commercially available 8.03 M solution of
methylamine in ethanol (Scheme 1). Starting from urea-Asp secondary amides, whatever alkyl 8a,
aryl 8b or α-amido ester 8c, cyclization occurred in less than five minutes at room temperature (rt)
giving rise the formation of DHO amides 9a-c, respectively, in almost quantitative yields (equations
1-3, Scheme 1). Moreover, starting from urea-Asp amide 8d bearing a methyl ester instead of benzyl
ester, after cyclization and work-up with 1N aqueous solution of HCl, product 9a was recovered very
clean without the need of a chromatographic purification (equation 4, Scheme 1). Quite surprisingly,
starting from urea-Asp tertiary amides 8e-g the cyclization reaction did not occur. Indeed, after
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stirring at rt for 12 hours, we obtained the formation of the corresponding N-methyl amides 10a,b,
arising from nucleophilic attack of methylamine to the Asp benzyl ester moiety, in the case of N,N-
dibenzyl and prolyl amides 8e,g, respectively, while decomposition of the starting material starting
from morpholine amide 8f (equations 5-7, Scheme 1).
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O
R1
O
R2
O
1
2
3
4
5
6
7
8
+
1-N3
N
N
=
R
R2-NCO
H
N
R3
3
4
Ph
N
CH C
3P,
3
9
R1
N
when R4
or
H
O
H
R3
NH
R4
MeNH2
EtOH
TMP,
H
3CN
C
N
R3
N R2
R1 N C
R2
N
R4
COOH
+
+
XOOC
O
H
N
XOOC
O
5
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9
R3
O
R2
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N
R4
N
N
R1
H
O
H
10
when R4
=
O
COOBn
O
N
NH2
CH3
H
N
H
N
Ph
1
eq.
Ph
N
H
N
CH3CN
N
O
O
<
rt
O
5',
8a
9a
O
COOBn
O
N
H
N
H
N
NH2
CH3
N
H
N
N
O
2
3
eq.
eq.
CH3CN
O
O
<
rt
5',
8b
9b
O
COOBn
O
N
H
N
NH2
CH3
H
N
COOBn
Ph
COOBn
Ph
N
H
N
N
O
CH3CN
O
O
<
rt
5',
8c
9c
O
COOMe
O
O
N
NH2
CH3
H
N
H
N
Ph
4
eq.
Ph
N
H
N
CH3CN
N
O
O
<
rt
O
O
5',
8d
9a
COOBn
N
N
O
H
N
NH2
CH3
Ph
Ph
N
H
N
N
N
5
6
eq.
eq.
H
CH3CN
Ph
O
Ph
O
rt
12h
,
8e
10a
COOBn
O
O
NH2
CH3
O
N
O
decomposition
N
H
N
CH3CN
rt
12h
,
8f
O
COOBn
N
NH
O
NH2
CH3
N
7
N
H
N
N
N
eq.
CH3CN
rt
H
COOBn
COOBn
O
O
12h
,
8g
10b
Scheme 1. Reaction of urea-Asp amides 8 with a solution of methylamine.
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After the observation that cyclization could be obtained in the same condition used for the MC
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process, namely using CH3CN as solvent, we explored the scope of this strategy for the sequential
MC synthesis of N,N’-disubstituted DHO amides. More in detail, azide 3 and isocyanate 4 were
mixed in CH3CN in the presence of Ph3P at room temperature (rt). Once carbodiimide 7 was formed
(TLC monitoring), the temperature was lowered to 0°C and amine 6, 2,4,6-trimethylpyridine (TMP)
followed by acid 5 were added and the solution stirred at rt for 3 hours. Once urea-Asp amide 8 is
formed, a commercial available solution of MeNH2 (8.03 M in ethanol) was added (10% in volume),
the solution stirred for 5 minutes and quenched with a 1N aqueous solution of HCl (Figure 3). We
started from a variety of azides 3{1-7} (chemset 1, Figure 2) and isocyanates 4{1-4} (chemset 2,
Figure 2), or available carbodiimides 7{a-b} (chemset 4, Figure 2), fumaric acid methyl and benzyl
monoesters 5{1-2} (chemset 3, Figure 2), and amines 6{1-8} (chemset 5, Figure 2). All the
corresponding final DHO amides were recovered in very good yields (Figure 3). Moreover, as
expected, the reaction was completely regioselective when asymmetric carbodiimides were used (see
mechanism in Scheme 3). Starting from chiral aminoacids as nucleophiles, a 1:1 distereoisomeric
mixture of DHO-containing dipeptides 9{a,1,5}, 9{a,1,6}, 9{1,1,2,5}, and 9{a,1,7} was obtained. It
is worth noting that compounds 9{a,1,7} could be considered a precursor for the synthesis of N,N’-
disubstituted analogues of Taltirelin, a thyrotropin-releasing hormone (TRH) analog containing DHO
acid moiety.16 The same 1:1 mixture of diastereoisomers 9{a,2,8} were obtained when lysine amide
6{8} was used as nucleophile. These results were expected since the chiral nucleophiles step in the
reaction when the new chiral stereocenter at the Asp moiety is already formed (see mechanism in
Scheme 3).
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The scope of this strategy was further extended to the synthesis of 4-Aph(Hor) conjugates 9 by
using 4-Aph derivatives 6{9-13} as nucleophiles (chemset 5, Figure 2). The first attempt using
commercially available DCC 7{b} lead to the formation of a 2:1 mixture of two inseparable products
(Scheme 2). Along with the desired DHO amide 9{b,2,9}, we obtained the formation of the side
product 11{b,2,9} arising from intramolecular attack of the aromatic amidic nitrogen instead of the
1
nitrogen belonging the urea moiety (stated from H NMR spectrum, Scheme S2). Thus, we turned
our attention in the optimization of the cyclization process starting from 4Aph-Asp-urea 8{b,2,9}
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isolated after the MC process. Among different conditions suitable with the use CH3CN as organic
solvent, we found that the addition of a 10% in volume of a 1N aqueous solution of NaOH produced
the regiospecific formation of DHO amide 9{b,2,9} in almost quantitative yields (Scheme 2).
Similarly, a 1:3 mixture in favor of the undesired derivatives 11{1,1,2,11} was obtained by
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3
4
5
6
7
8
9
1
cyclization triggered by methylamine solution of intermediate 8{1,1,2,11} (stated from H NMR
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spectrum, Scheme S3) that was obtained from the MC process with asymmetric carbodiimide
7{1,1}, whereas regiospecific cyclization leading to the formation of target compound 9{1,1,2,11}
was triggered by using 10% in volume of a 1N aqueous solution of NaOH (Scheme 2).
-
Azides
Chemset 1
N3
N3
O
N3
N3
BocHN
N3
N3
N3
H
3CO
O
3
3
3
3
3
3
3
{7}
{1}
{2}
{3}
{4}
{5}
{6}
-
4
Chemset
Carbodiimides
-
-
monoesters
Chemset 2
Chemset 3 Fumaric acid
Isocyanates
N
N
C
NCO
COOH
COOH
7
NCO
BnOOC
{a}
NCO
{1}
MeOOC
NCO
5
5
{2}
{1}
N
N
C
4
4
4
4
{4}
{2}
{3}
7
{b}
-
Amines
Chemset 5
NH2
Ph
N
Ph
Ph
N
Ph
O
NH2
H
N
NH2
NH2
H2N
H2N
COOBn
{6}
H2N
COOBn
{7}
N
H2N
BocHN
COOBn
{5}
H
O
6
6
6
6
{8}
6
6
6
{3}
6
{4}
{1}
{2}
NH2
NH2
NH2
NH2
NH2
O
O
O
H
H
N
H
BocHN
Ph
N
N
O
O
N
BocHN
BocHN
AcHN
BocHN
H
O
O
O
O
O
6
6
6
6
6
{13}
{9}
{10}
{11}
{12}
Figure 2. Selected chemsets for the synthesis of the library of N,N’-disubstituted DHO amides:
azides (chemset 1), isocyanates (chemset 2), fumaric acid monoesters (chemset 3), commercial
available carbodiimides (chemset 4), and amines (chemset 5).
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+
1-N3
R
R2-NCO
1
2
3
4
5
6
7
3
4
Ph
N
CH C
3P,
3
O
R1
N
MeNH2
Method A
R1
O
R2
O
O
H
R3
NH
R4
TMP,
H
C
3CN
N
N
N
R3
H
N R2
R1 N C
R2
N
R4
COOH
+
+
N
XOOC
or
R3
O
1N NaOH
Method B
XOOC
7
5
6
9
8
8
9
OCH3
10
11
12
13
14
15
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t
O
O
BuOOC
O
O
O
N
N
O
N
N
N
N
O
N
N
O
O
O
N
N
O
O
O
NH
NH
NH
O
O
NH
NH
9
9
9
{a,1,2} (88%)
Method A
9
{3,1,2,2} (62%)
{7,1,2,3} (63%)
Method A
{a,1,1} (93%)
Method
A
Method A
9
{1,1,2,2} (68%)
Method A
OCH3
t
BuOOC
O
O
O
O
O
N
N
N
O
N
N
N
N
N
O
O
O
O
O
O
N
N
O
NH
N
NH
NH
COOCH2
NH
COOCH2
O
Ph
O
tBu
COO
N
HN
Ph
O
Ph
NH
COOCH2
Ph
Ph
9
{a,1,7} (76%)
Method A
Ph
Ph
9
9
9
{a,1,6} (84%)
Method A
{3,1,2,4} (55%)
Method A
{a,1,5} (91%)
Method A
9
{1,1,2,5} (69%)
Method A
OMe
O
O
O
O
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
O
N
N
O
NH
NH
NH
NH
O
O
NH
H
N
BocHN
BocHN
COOEt
BocHN
COOEt
BocHN
COOEt
BocHN
COOEt
9
9
9
{2,1,1,9} (64%)
{a,2,9} (88%)
Method B
{b,2,9} (82%)
Method B
9
{a,2,8} (87%)
Method A
Method B
9
{1,1,1,9} (61%)
Method B
O
O
O
O
O
O
O
BocHN
N
N
O
N
N
N
N
N
N
O
O
O
O
O
O
NH
NH
NH
NH
BocHN
COOEt
BocHN
COOEt
BocHN
COOEt
BocHN
COOEt
9
{4,1,1,9} (61%)
Method
9
{6,1,2,9} (57%)
Method B
9
9
{3,1,1,9} (72%)
Method B
B
{5,3,1,9} (66%)
Method B
O
O
O
O
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
OMe
NH
NH
NH
NH
H
N
BocHN
AcHN
COOEt
BocHN
COOEt
BocHN
COOEt
O
9
9
9
{a,1,10} (71%)
Method B
9
{6,2,2,9} (62%)
Method B
{1,4,1,9} (51%)
Method B
{a,1,11} (82%)
Method B
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OMe
1
O
O
2
3
4
5
6
7
8
9
O
N
N
N
N
O
N
N
O
O
N
N
O
O
O
O
NH
NH
O
O
NH
NH
O
H
H
N
N
H
BocHN
O
BocHN
N
H
BocHN
N
O
O
BocHN
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
9
9
{7,1,1,11} (66%)
{b,1,12} (88%)
Method B
9
{5,1,1,11} (69%)
Method B
B
Method
9
{1,1,2,11} (62%)
Method B
OMe
O
N
N
O
O
O
N
N
NH
O
O
O
NH
H
N
BocHN
N
H
O
O
O
H
N
BocHN
O
9
{7,1,2,13} (53%)
Method B
9
{1,1,2,12} (69%)
Method B
Figure 3. Library of N,N’-disubstituted DHO amides encompassing 4-Aph(Hor) derivatives.
O
O
N
N
N
O
O
MeNH2
HN
O
NH
<
rt, 5'
N
CH3CN,
+
O
O
N
N
H
BocHN
COOEt
BocHN
11
COOEt
{b,2,9} (31%)
O
9
{b,2,9} (62%)
NH
COOBn
{b,2,9}
O
8
BocHN
COOEt
N
N
O
O
1N NaOH
NH
<
rt, 5'
CH3CN,
9
{b,2,9} (92%)
BocHN
COOEt
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ACS Combinatorial Science
OMe
OMe
1
2
3
4
5
O
O
N
N
N
O
O
HN
OMe
O
MeNH2
6
7
8
9
NH
N
<
rt, 5'
CH3CN,
+
O
O
H
H
N
N
N
N
BocHN
BocHN
H
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
11
{1,1,2,11} (63%)
9
NH
COOBn
{1,1,2,11} (21%)
OMe
H
N
BocHN
O
O
8
{1,1,2,11}
1N NaOH
N
N
O
<
rt, 5'
CH3CN,
O
NH
H
9
N
{1,1,2,11} (95%)
BocHN
O
Scheme 2. Attempts to regioselective cyclization of 4-Aph-Asp-urea 8.
With this result in hand, we checked the possibility to apply these conditions for the MC sequential
synthesis of 4-Aph(Hor) conjugates 9. We were delighted to observe that in all cases the formation
of desired conjugates 9 arose with good yields through a one pot, completely regiospecific
cyclization process, i.e. we did not detect the formation of byproducts 11 in any reaction performed.
Indeed, the reaction worked efficiently starting either from 4-Aph esters with different protecting
groups at the α-nitrogen, such as Nα-Boc-4-Aph ethyl ester 6{9}, and Nα-Ac-4-Aph ethyl ester
6{10}, from 4-Aph amide 6{11}, and from 4-Aph dipeptides 6{12-13} (chemset 5, Figure 2). Again,
it was not surprising that starting from azides and isocyanates with different substituents, the reaction
was totally regiospecific leading to the formation of DHO derivatives having the less sterically
hindered substituent (or aromatic substituent as for compound 9{1,5,1,9}) at the N-3 position. Quite
surprisingly, the diastereosisomers formed during the reactions with 4-Aph derivatives 6{9-13} are
isochrones in all cases. Indeed, although a 1:1 mixture of two diastereoisomers is formed, the 1H and
13C NMR spectra of the inseparable mixtures are very clean showing a single set of signals (see
copies of the NMR spectra in SI). In order to prove the presence of the two diastereoisomers, we
recovered intermediate 8{5,1,2,10} and submitted to hydrogenolysis of the benzyl ester (which
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cleaved also the benzyl moiety at N-3) followed by coupling with (S)-alanine methyl ester (Scheme
S4). NMR spectra of the final adduct 14 showed two sets of signals belonging to the two
diasterosisomers (see 1H and 13C NMR in SI).17
1
2
3
4
5
6
The currently accepted mechanism of the sequential MC process18 starts with reaction between
azide 3 and isocyanate 4 in the presence of Ph3P that produces carbodiimide 7 through
Staudinger/aza-Wittig reactions (Scheme 3).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R
1 N3
R2
N
COOH
R1
N
XOOC
3
+
R1
Ph3P
5
aza-Michael
R1
N
N
R2
XOOC
O
N
C
O
R3-NH2
H
7
XOOC
R2
N
R2
O
NCO
12
Ph3PO
6
O
H2N-R3
4
13
R1
alkyl R
tertiary alkyl
=
2 = secondary or
or
;
primary
R3-NH2 alkyl amine
=
O
COOX
amine
2
1 = secondary
=
aryl
α
R2
N
R1
O
R
alkyl R
tertiary alkyl
;
O
MeNH2
H
N
N
-aminoester
or
R2
H
N
H
N
R1
R3
N
2
1 = aromatic;
=
R3
R
X
R
alkyl
O
O
9
=
or
8
Me Bn
O
R2
HN
O
O
R2
N
H
N
N
R1
N
O
N
R1
O
+
O
MeNH2
COOX
H
N
O
9
11
N
R2
N
R3-NH2 4-Aph derivatives
=
H
H
O
N
H
N
O
O
R1
R2
O
O
N
8
H
N
N
H
1M NaOH
N
R1
O
O
9
N
H
O
Scheme 3. Proposed mechanism of the sequential MC process
It is worth noting that this is the yield-limiting step since when we run the sequential MC/post-
cyclization process with commercially available carbodiimides 7{a,b}, the final adducts are obtained
with excellent yields in all cases. Once carbodiimide 7 is formed (TLC monitoring), the addition in
the reaction solution of amine 6 followed by acid 5 leads to the formation of O-acylisourea
intermediate 12 which readily cyclizes to 13 before the amine can acts as nucleophile as it happens in
usual carbodiimide-promoted coupling reactions. The cyclization process is totally regiospecific and
arises from nucleophilic attack of the less hindered nitrogen. Once 13 has formed, the amine steps-in
the reaction mechanism leading to the formation of urea-Asp amide 8. The addition of a base triggers
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the immediate cyclization of urea-Asp secondary amides to afford DHO amides 9, while starting
from urea-Asp tertiary amides the cyclization process did not occur. In general, MeNH2 works
efficiently. However, when the amidic proton of 8 is more acidic, such as in DHO amide arising
from the nucleophilic attack of 4-Aph derivatives 6{9-13}, cyclization leading to the formation of
succinimide ring 11 becomes competitive. In these cases, the use of a stronger base such as a 1N
aqueous solution of NaOH is required to obtain a regiospecific cyclization.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To explain these results, we first reasoned that some conformational effects, due to hydrogen bond
networking, could be responsible for different reactivity of the urea-NH nitrogen in affording product
9 or 10.15 Firstly, we computationally investigated the conformational behavior of compounds 8a,b,d
and 8e to gather information about the presence of hydrogen bonds involving the urea-NH hydrogen,
thus inhibiting the reactivity toward the intramolecular cyclization. In compound 8a,b,d two NH (a
urea-NH and an amide-NH) are present which are capable of forming four distinct hydrogen bonds
with carbonyls: A and B H-bonds involve the urea moiety whereas C and D H-bonds belong to the
amide bond (A-D, Figure 4). For compound 8e only the urea-NH can be involved in two distinct H-
bond (A, B, Figure 4)
A
C
O
O
NH
=R
8a
8b
8d
8e
R1=H, R2 3=Bn
O
R1=H, R2=Ph, R3=Bn
R1=H, R2=Bn, R3=Me
=R2=R
H
OR3
R1
H
N
N
N
N
R2
N
R1
3=Bn
O
O
R2
OR3
D
B
O
Figure 4. Intramolecular hydrogen bond patterns for compounds 8a,b,d,e.
A conformational analysis was performed with a combined Monte Carlo search and a Molecular
Mechanics energy minimization (MM/MC). Results are reported in Table 1 as percentage of
conformers, which have a specific intramolecular hydrogen bond.
Table 1. Results from conformational analysis.
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Urea NH H-Bond
Amide NH H-Bond
1
2
3
4
5
6
7
Compound
A
B
C
D
12%
5%
27%
--
4%
3%
8%
36%
17%
5%
25%
5%
27%
61%
22%
--
8a
8b
8d
8e
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
From this analysis, it appears that for compounds 8a,b,d there is a preference to form an
intramolecular H-bond C and D with the amide-NH (39% of conformers for 8a, 66% for 8b and 49%
for 8d) over the urea-NH hydrogen. As obvious, for 8e only the urea-NH can be considered to form
an H-bond (41% of conformers). For compound 8a we also compared the lowest energy conformers
having the urea-NH or the amide-NH hydrogen bonds by computing their energy by DFT
calculations at the 6-31G(d,p) level and results confirmed that there is a preference for the formation
of a type C intramolecular hydrogen bond (Figure 5).
Figure 5. Lowest energy conformer for 8a (left, rel. E = 0.00 Kcal/mol) and the first conformer
having an urea-NH hydrogen bond (right, rel. E = 0.42 Kcal/mol).
These observations prompted us to suppose that the presence of an intramolecular hydrogen bond
with the urea-NH in compound 8e locked the nitrogen atom in a less reactive conformation and this
could be one of the reasons why the intramolecular cyclization cannot occur. Moreover, this H bond
can activate the ester function toward nucleophilic attack, thus favoring the formation of amide
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product 10. For compounds 8a,b,d, the urea-NH nitrogen is free to react because of a preference to
form the intramolecular H-bond with the amide moiety and the cyclization to form 9a,b,d can take
place. Then, we turned our attention to the possible importance of the second substituent on the urea
nitrogen not involved in the cyclization process.19 Indeed, a preliminary study on the acidity of the
NH groups revealed that the urea-NH becomes much more acidic (four order of magnitude) when the
other nitrogen belonging the urea moiety is disubstituted, becoming almost two order of magnitude
more acidic than the amide-NH (Figure 6).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
COOBn
COOBn
COOBn
N
O
O
O
H
N
H
N
Ph
Ph
Ph
N
H
N
N
H
N
N
H
N
H
Ph
O
O
O
15.84 +/- 0.46
0.46
15.27 +/- 0.46
17.59
0.70
+/-
13.55 +/- 0.46
13.55 +/- 0.46
12.16
+/-
Figure 6. pKa values of the NH moieties. pKa values were calculated with the software ACD Labs
6.0
Thus, we synthetized urea-Asp conjugate 15 through a stepwise strategy, namely coupling Boc-L-
aspartic acid 4-benzyl ester 14 with pentyl amine followed by N-deprotection and reaction with
adamantly isocyanate (lipophilic admantyl and pentyl substituents were chosen to make compound
15 soluble in acetonitrile). Compound 15 was dissolved in CH3CN and treated with a 10% in volume
of a 8.03 M solution of methylamine in ethanol. Under these conditions we slowly obtained (12
hours) the five-member imide ring 16 which proved to be very stable (Scheme 4).20 This result
clearly demonstrates that in order to obtain the fast cyclization to DHO amides 9, it is mandatory that
the urea nitrogen not involved in the process is disubstituted.21
O
Ph
COOCH2
H
N
EDC, DCM
isocyanate,
1) pentylamine,
2)
Ph
COOCH2
O
NH2
CH3
TFA,
DCM
O
N
N
N
adamantyl
CH3CN
12 h
3)
N
N
H
BocHN
COOH
H
H
H
TEA,
O
DCM
O
14
15
three steps)
16
(58%,
(75%)
Scheme 4. Cyclization of disubstituted urea-Asp 15.
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1
2
3
4
5
6
7
8
9
Based on the experimental and computational results, and on literature reports, a possible
mechanism for the cyclization process is shown in Scheme 5. We speculate that, when R1 ≠ H and R4
= H, deprotonation of the more acidic urea-NH triggers the formation of a formal negative charge on
the amide oxygen which attacks the ester moiety generating a highly reactive 5-membered lactone-
like intermediate A.22 In the next step, A can undergo opening by the attack of nucleophilic urea
nitrogen producing final DHO amide 9. It is worth noting that these two processes are very fast,
occurring in less than five minutes. Starting from tertiary amides (R1 and R4 ≠ H) this mechanism
cannot occur. In this case, the ester moiety, activated by the intramolecular hydrogen bond, slowly
reacts with the nucleophilic base giving rise the formation of the methyl amide. Finally, starting from
N,N’-monosubstituted urea-Asp conjugates (R1 and R4 = H) deprotonation occurs at the more acidic
NH-amide producing the slow formation of stable five-membered imide ring 18 when the base is
methylamine, while hydrolysis of the ester moiety occurs when the base is aqueous NaOH.19
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
O
X
R1
N
R2
O
R1
O
O
O
R1
R4
H
H
R1
R2
N
=
=
O
N
N
H
B:
H
N
R2
N
N
R3
N
O
N
R3
R
O
H
H
9
A
O
:B
O
COOX
R3
R2
H
X
B
O
O
R1
R4
H
H
B:
N
=
=
O
N
R3
R2
N
R2
N
R3
R4
N
N
R1
R4
N
H
N
R1
R4
H
N
R1
O
O
O
O
8
10
O
O
N
X
R3
O
R1
R4
H
H
O
O
N
=
=
R3
R2
R2
N
N
N
N
:B
H
H
H
H
H
O
O
18
Scheme 5. Proposed mechanism for the cyclization process.
CONCLUSION
In conclusion, we have developed for the first time a sequential MC domino process for the
synthesis of intriguing DHO amides by a one-pot, post-cyclization of N-urea-aspartate which arises
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under mild conditions. The process is high yielding and, by selecting the suitable base to trigger the
last cyclization step, totally regioselective. The process is also modular since it works efficiently with
different alkyl and aromatic azides and isocyanates, fumaric acid monoesters and primary amines. In
particular, starting from α-aminoacid or peptides as nucleophiles it is possible to prepare libraries of
peptidomimetics having at the N-terminus a N,N’-disubstituted DHO heterocycle. Moreover, by
running the process in the presence of 4-Aph derivatives, we were able to prepare a large array of
structurally diverse conjugates bearing N-substituted 4-Aph(Hor) moiety which is a key component
of GnRH antagonists such as Degarelix. The application of the described methodology, which looks
also suitable for solid-phase/combinatorial synthesis, to incorporate N-substituted DHO amides into
known DHO-containing biologically relevant compounds or to find new molecular hits, as well as
the development of a stereoselective version of the process, is currently in progress in our
laboratories.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental Section
General Methods: Commercially available reagent-grade solvents were employed without
purification. TLC were run on silica gel 60 F254 Merck. Flash chromatography (FC) was performed
with silica gel 60 (60-200 µm, Merck). 1H NMR spectra were recorded on 400 MHz spectrometers.
Chemical shifts are expressed in ppm (δ), using tetramethylsilane (TMS) as internal standard for 1H
and 13C nuclei (δH and δC = 0.00). 4-Aph derivatives 6{9-13} were prepared starting from
commercial available Boc-4-nitro-L-phenylalanine with usual coupling reaction followed by
hydrogenation at ambient pressure catalyzed by Pd/C of the nitro group. Compound 8a,g were
obtained as described in reference 10 b.
General procedure for the MC process followed by post-cyclization with a solution of
MeNH2 in ethanol. To a solution of para-methoxybenzyl azide 3{1} (0.31 mmol, 1 equiv) and tert-
butyl isocyanate 4{1} (0.32 mmol, 1.05 equiv) in CH3CN (3 mL), solid Ph3P (0.32 mmol, 1.05
equiv) was added at rt. The solution is left to stir overnight. The temperature is lowered to 0 °C and
TMP (0.32 mmol, 1.05 equiv), neat iso-butyl amine 6{2} (0.32 mmol, 1.05 equiv) followed by a
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solution of acid 5{2} (0.26 mmol, 1.2 equiv) in a minimum amount of CH3CN were added. The
reaction was left to stir at rt until complete disappearance of the carbodiimide was detected (TLC
monitoring, typically 3 hours). A commercial available 8.03 M ethanolic solution of MeNH2 (0.3
mL) was added at rt. After 5 minutes the reaction was acidified with a 1N aqueous solution of HCl
and extracted with AcOEt. The collected organic phases were dried over Na2SO4, filtered, and the
solvent evaporated. The crude was purified by flash chromatography affording compound 9{1,1,2,2}
in 68% isolated yield.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Representative example of DHO acid derivative. 1-(tert-butyl)-N-isobutyl-3-(4-
methoxybenzyl)-2,6-dioxohexahydropyrimidine-4-carboxamide 9{1,1,2,2}: Rf = 0.37 (hexane:AcOEt
= 50:50); 1H-NMR (400 MHz, CDCl3): δ = 7.21 (d, J = 8.8 Hz, 2H, aromatics), 6.84 (d, J = 8.8 Hz,
2H, aromatics), 5.83 (br s, 1H, NH), 4.72 (d, J = 14.8 Hz, 1H), 4.21 (d, J = 14.8 Hz, 1H), 3.98 (t, J =
5.2 Hz, 1H), 3.78 (s, 3H), 3.02-2.98 (m, 2H), 2.62 (dd, J = 15.2 and 5.2 Hz, 1H), 2.51 (dd, J = 15.2
13
and 5.2 Hz, 1H), 1.70 (septet, J = 6.4 Hz, 1H), 1.61 (s, 9H), 0.87 (d, J = 6.4 Hz, 6H); C-NMR
(100.6 MHz, CDCl3); δ = 173.7, 168.1, 159.2, 157.3, 129.6, 128.4, 114.1, 58.1, 55.6, 55.2, 47.1,
44.5, 36.8, 28.7, 28.3, 20.09, 20.08; ESI (m/z) 412.2, [M+Na, (100)]+; Anal. calcd. for C21H31N3O4:
C 64.76, H 8.02, N 10.79; found: C 64.77, H 8.02, N 10.80.
General procedure for the MC process followed by post-cyclization with 1M aqueous
solution of NaOH. To a solution of para-methoxybenzyl azide 3{1} (0.31 mmol, 1 equiv) and tert-
butyl isocyanate 4{1} (0.32 mmol, 1.05 equiv) in CH3CN (3 mL), solid Ph3P (0.32 mmol, 1.05
equiv) was added at rt. The solution is left to stir overnight. The temperature is lowered to 0 °C and
TMP (0.32 mmol, 1.05 equiv), a solution of amine 6{9} (0.32 mmol, 1.05 equiv) in a minimum
amount of CH3CN followed by a solution of acid 5{1} (0.26 mmol, 1.2 equiv) in a minimum amount
of CH3CN were added. The reaction was left to stir at rt until complete disappearance of the
carbodiimide was detected (TLC monitoring, typically 3 hours). A 1N aqueous solution of NaOH
(0.3 mL) was added at rt. After 5 minutes the reaction was acidified with a 1N aqueous solution of
HCl and extracted with AcOEt. The collected organic phases were dried over Na2SO4, filtered, and
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the solvent evaporated. The crude was purified by flash chromatography affording compound 9
1
2
3
{1,1,1,9} in 61% isolated yield.
4
5
Representative
example
of
4-Aph(Hor)
derivative.
ethyl
(2S)-2-((tert-
6
7
8
butoxycarbonyl)amino)-3-(4-(1-(tert-butyl)-3-(4-methoxybenzyl)-2,6-dioxohexahydropyrimidine-4-
9
1
carboxamido)phenyl)propanoate 9{1,1,1,9}. Rf = 0.35 (hexane:AcOEt = 40:60); H-NMR (400
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MHz, CDCl3): δ = 7.74 (br s, 1H, NH-amide), 7.28 (d, J = 8.0 Hz, 2H, aromatics), 7.12 (d, J = 8.4
Hz, 2H, aromatics), 6.99 (d, J = 8.4 Hz, 2H, aromatics), 6.71 (d, J = 8.0 Hz, 2H, aromatics), 4.90 (br
d, J = 7.6 Hz, 1H, NH-carbamate), 4.57 (d, J = 15.2 Hz, 1H), 4.45 (m, 1H), 4.30 (d, J = 15.2 Hz,
1H), 4.08 (q, J = 7.2 Hz, 2H), 3.96 (m, 1H), 3.65 (s, 3H), 3.00 (dd, J = 11.6 and 5.6 Hz, 1H), 2.94
(dd, J = 11.6 and 5.6 Hz, 1H), 2.63 (m, 2H), 1.55 (s, 9H), 1.35 (s, 9H), 1.17 (t, J = 7.2 Hz, 3H); 13C-
NMR (100.6 MHz, CDCl3); δ = 173.9, 171.7, 166.1, 159.3, 157.2, 136.5, 132.2, 129.9, 129.7, 128.6,
128.3, 119.7, 114.2, 79.9, 61.4, 58.3, 55.8, 55.2, 54.5, 44.7, 37.9, 37.7, 28.7, 28.3, 14.2; ESI (m/z)
663.1 [M+K, (5)]+, 647.2, [M+Na, (100)]+; Anal. calcd. for C33H44N4O8: C 63.44, H 7.10, N 8.97;
found: C 63.42, H 7.10, N 8.98.
Acknowledgments. Politecnico di Milano is gratefully acknowledged for economic support.
Abbreviations
DHO, dihydroorotic; DHODH, dihydroorotate dehydrogenase; GnRH, gonadotropin-releasing
hormone; 4-Aph, 4-aminophenylalanine; 4-Aph(Hor), N4-(dihydroorotyl)-4-aminophenylalanine;
Asp, aspartic.
Supporting Information Available: Characterization of all the new compounds not present in the
1
13
Experimental Section. Copies of H NMR, C NMR, and ESI-MS spectra of all new compounds.
This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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Corresponding Author
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*Email: alessandro.volonterio@polimi.it.
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ORCID
Alessandro Volonterio: https://orcid.org/0000-0002-0125-0744
References
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dihydroorotate dehydrogenase as a therapeutic target for cancer. Pharmacol. Ther. 2019, 195, 111-
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system actions of thyrotropin-releasing hormone analogues containing a dihydroorotic acid moiety. J.
Med. Chem. 1990, 33, 2130-2137.
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pure 1- and 3-substituted dihydroorotic acids. Chem. Pharm. Bull. 1989, 37, 1764-1769.
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lane towards molecular complexity. Chem. Eur. J. 2008, 14, 8444-8454; (b) Dömling, A.; Wang, W.;
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sequential synthesis of hydantoins. Org. Biomol. Chem. 2012, 10, 9538-9555; (b) Bellucci, M. C.;
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Terraneo, G.; Volonterio, A. Multi-component synthesis of peptide-sugar conjugates. Org. Biomol.
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Multicomponent synthesis of N-carbamoyl hydantoin derivatives. Synlett 2013, 24, 727-732; (d)
Bellucci, M. C.; Volonterio, A. Synthesis of N-glycosyl conjugates through a multicomponent
domino process. Eur. J. Org. Chem. 2014, 2386-2397.
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approach of Passerini reaction: one-pot synthesis of β-acyloxyamides and phtalides. Org. Lett. 2017,
19, 4616-4619; (c) Martinez-Ariza, G.; Ayaz, M.; Medda, F.; Hulme, C. Synthesis of diverse
nitrogen-enriched heterocyclic scaffolds using a suite of tunable one-pot multicomponent reactions.
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diversity, and complexity with multicomponent reactions. Synlett, 2013, 24, 666-685.
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15. In literature we found only one example for the synthesis of DHO acid amides by cyclization of
ureido-Asp cyclic amides: Herrero, S., Salgado, A., Garcia-Lopez, M. T., Herranz, R. Synthesis of
chiral 1,6,8-trioxoperhydropyrazino[1,2-c]pyrimidines as novel highly functionalized scaffolds for
peptidomimetics. Terahedron Lett. 2002, 43, 4899-4902. In this work, the cyclization process was
influenced by the stereochemistry of tryptophan of urea-oxopiperazine derivatives. Indeed,
cyclization of compounds bearing L-tryptophan arose with an equimolar amount of DBU at rt, while
starting from the analogues bearing D-tryptophan it was necessary to heat at refluxing temperature.
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16. Kinoshita, K., Yamamura, M., Sugihara, J., Suzuki, M., and Matsuoka, Y. Taltirelin hydrate
(TA-0910): an orally active thyrotropin-releasing hormone mimetic agent with multiple actions. CNS
Drug Rev. 1998, 4, 25–41.
17. We tried to perform HPLC analysis on different compounds 9. We were able to obtain good
separation only with compound 9{a,2,9} which chromatogram showed the presence of two peaks
with an area ratio of 1:1 (see SI, page S67).
18. Marcelli, T., Olimpieri, F., Volonterio, A. Domino synthesis of 1,3,5-trisubstituted hydantoins: a
DFT study. Org. Biomol. Chem. 2011, 9, 5156-5161.
19. Looking at the literature, we found only one example where treatment of N-aryl-N’-urea-Asp
amide 4-benzyl ester with a 1N aqueous solution of NaOH lead to the hydrolysis of the ester function
rather than cyclization to DHO amide:
Ph
COOCH2
COOH
Cl
Cl
O
O
H
N
H
N
NaOH
N
N
N
N
H
H
H
H
H
2O/MeOH
O
O
N
N
N
N
Song, Y.; Zhu, B.; Wang, S.; Bhakta, C.; Scarborough. Preparation of amino acid urea derivatives as
factor Xa inhibitors. 2006, WO 2006063113.
1
1
20. The structure of compound 16 was determined through H and H-1H COSY NMR (see SI,
Scheme S5). The spectra clearly show the presence of two signals belonging to NH protons: one
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singlet resonating at 5.38 ppm (urea-NH linked to adamantane) and one doublet (other urea-NH)
resonating around 6.24 ppm, which shows a cross peak with a multiplet resonating around 4.26 ppm
belonging to the Cα of the Asp moiety.
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9
21. A similar attempt was made by adding a 10% in volume of a 1N aqueous solution of NaOH to 4-
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Aph-Asp derivative 17 dissolved in CH3CN:
BnOOC
O
H
N
N
H
N
H
O
1N
NaOH
mixture unidentified
of
products
CH3CN
17
AcHN
COOEt
However, in this case we obtained a mixture of unidentified products.
22. In hope of trapping a reactive intermediate, we performed the cyclization of 4-Aph-Asp
8{b,2,9} in the presence of 1, 5, and 10 equivalents of highly electrophilic benzyl bromide. However,
by quenching the reaction after 5 minutes, we always recovered 4-Aph(Hor) 9{b,2,9} as the only
product.
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