An access to a library of novel triterpene derivatives with a promising pharmacological potential by Ugi and Passerini multicomponent reactions

Accepted Manuscript An access to a library of novel triterpene derivatives with a promising pharmacological potential by Ugi and Passerini multicomponent reactions Jana Wiemann, Lucie Heller, René Csuk PII:

S0223-5234(18)30197-1

DOI:

10.1016/j.ejmech.2018.02.060

Reference:

EJMECH 10237

To appear in:

European Journal of Medicinal Chemistry

Received Date: 6 December 2017 Revised Date:

16 February 2018

Accepted Date: 18 February 2018

Please cite this article as: J. Wiemann, L. Heller, René. Csuk, An access to a library of novel triterpene derivatives with a promising pharmacological potential by Ugi and Passerini multicomponent reactions, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.060. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT An access to a library of novel triterpene derivatives with a promising pharmacological potential by Ugi and Passerini multicomponent reactions

Jana Wiemann, Lucie Heller, René Csuk* Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str. 2, D-

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06120 Halle (Saale), Germany

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Graphical abstract

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Abstract

The promising combination of natural product leads and their derivatization by isocyanidebased multicomponent reactions (IMCRs) has gained interest in accessing diversity-oriented libraries with auspicious pharmacological potential. Therefore, a set of 34 Ugi and 3 Passerini

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products was successfully synthesized starting from naturally occurring triterpenoids, i.e. oleanolic acid (OA) and maslinic acid (MA), followed by a biological evaluation of the novel α-acylamino carboxamides and the α-acyloxy carboxamides in colorimetric SRB assays to

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determine their cytotoxic potential. Especially, the MA-Ugi products 6a, 6b and 7b showed a remarkable cytotoxicity for A2780 ovarian carcinoma cells in a low µM range. Compounds 6a and 7b induced programmed cell death in part through the apoptosis pathway.

1. Introduction Natural products are quite often an inspiration for the development of new drugs [1]. As a result in the last 30 years about 50% of the approved small molecule drugs were natural based or natural inspired [2]. The significance of terpenes, particularly of triterpenes, for developing new drugs, however, was limited although they are an important class of natural products. They have been investigated intensely concerning their inherent biological activity. Their 1

ACCEPTED MANUSCRIPT antibacterial [3], antiviral [4, 5], antioxidative [6], anti-inflammatory [7, 8] and especially their cytotoxic properties [7, 9, 10] showed their significance as potential drug leads. Systematic modifications to optimize the biological activity of leads are among the most timeconsuming and cost-intensive steps in the development of new drugs. While there are several strategies for efficient activity based structural modifications, a diversity-oriented synthesis is

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particularly profitable and most rewarding. In this context, the manifold applications of multicomponent reactions (MCRs) [11-17] in medicinal chemistry have attained interest; they provide an easy and powerful access to diversity oriented libraries. Particularly, modifications of natural product leads by MCRs seem suited to afford substances with a promising

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pharmacological potential.

The term MCRs (multicomponent reactions) comprises reactions of three or more (up to eight [18]) components in a one-pot procedure yielding one final product with high atom economy.

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Experiments by Laurent and Gerhardt [19] performed as early as 1838 describing the reaction of bitter almond oil with ammonia mark the actual beginning of the MCRs. However, the Strecker synthesis (S-3CR) [20] of α-amino acids, published 12 years later, is considered to be the first official publication holding the concept of MCRs in preparative chemistry. In 1958 isocyanides became generally available from formylamines [21] thus opening an access

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to a multitude of new reactions: isocyanide-based multicomponent reactions (IMCRs), including the three component Passerini reaction (P-3CR, 1921) [22] and the four component Ugi reaction (U-4CR, 1959) [23]. Nowadays, the U-4CR is the most investigated and applied MCR at all. Based on the variability of the used components (amine, carbonyl compound,

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carboxylic acid and isocyanide) the synthesized α-acylamino carboxamides show a high degree of molecular product diversity, thus emphasizing the versatile application in combinatorial chemistry and especially in drug discovery [e.g. successful syntheses of the

[25].

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HIV inhibitor indinavir (CrixivanTM) [24] and the local anesthetic lidocaine (XylocainTM)]

Thus, we focused on the development of a library of triterpene derivatives obtained from IMCRs, especially by the U-4CR. The U-4CR-products are of special interest because they hold a dipeptide moiety. Prior investigations showed triterpenoid derived amides to be highly cytotoxic [26-29]. Therefore, we synthesized 37 triterpene derivatives by MCRs and determined their cytotoxicity in colorimetric SRB assays.

2. Results and Discussion

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ACCEPTED MANUSCRIPT A small library of derivatives was obtained by the U-4CR and the P-3CR (Fig. 1); an overview of the used components is shown in Fig. 1. Although there are numerous reports on IMCRs the number of references covering IMCRs and triterpenes is restricted to two examples [30, 31]. Thereby glycyrrhetinic acid was modified by U-4CR reactions [30] and several triterpenoids were subject of Passerini reactions [31] but no biological evaluations of

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the products have been reported. Our own research was focused to modify pentacyclic

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triterpenoids by IMCRs to increase the cytotoxicity of the parent compounds.

Fig. 1. Starting materials for the derivatization of triterpenoic acids by Ugi-4CR and by

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Passerini 3-CR.

At first, we investigated the U-4CR derivatization of oleanolic acid (OA). In general, U-4CRs are carried out in polar-aprotic solvents using high concentrations of the components [32].

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Because of the low solubility of the triterpenoic acids in these solvents, our reactions were characterized by long reaction times and moderate conversions. Increasing the reaction temperature and the use of microwave irradiation, however, did not lead to higher yields but the reaction time could be reduced to 2 days, and unchanged starting material was recovered. A first set of Ugi products 1a-1j was obtained from tert-butyl isocyanide; the amino component was varied to examine the effect of the substituent R2 on the cytotoxicity of the compounds (Scheme 1).

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Scheme 1. Synthesis of OA-Ugi products 1a-1j: a. MeOH, rt, 7–12 days, 59–80%; b. Ac2O, pyridine, rt, 12h, 87–95%.

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The biological activity of these compounds was examined in colorimetric SRB assays, and the EC50 values were determined for six human tumor cell lines and nonmalignant mouse fibroblasts. The results are compiled in Table 1.

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Table 1. Cytotoxicity of compounds 1a-1j and oleanolic acid (OA, for comparison); EC50 values in µM from SRB assay after 96 h of treatment; the values are averaged from three

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independent experiments performed each in triplicate; confidence interval CI = 95%; n.d. not detected/not determined. Human cancer cell lines: 518A2 (melanoma), A2780 (ovarian carcinoma), HT29 (colorectal carcinoma), MCF7 (breast carcinoma), A549 (lung adenocarcinoma), FaDu (hypopharyngeal carcinoma with multidrug resistance) and nonmalignant mouse fibroblasts (NIH 3T3).

EC50

R1

1a

H

R2

518A2

A2780

HT29

MCF7

A549

FaDu

NIH 3T3

7.9 ± 0.5

11.0 ± 0.8

13.8 ± 0.6

12.2 ± 0.6

8.6 ± 0.3

8.1 ± 0.3

11.2 ± 1.0

4

ACCEPTED MANUSCRIPT Ac

8.8 ± 0.6

4.1 ± 0.1

23.7 ± 2.7

5.7 ± 0.2

n.d.

6.7 ± 0.4

9.9 ± 0.8

1c

H

24.4 ± 3.1

10.5 ± 1.3

9.9 ±0.9

7.0 ± 1.1

27.7 ± 3.2

26.0 ± 4.8

15.5 ± 1.9

1d

Ac

12.5 ± 1.6

>30

12.1 ± 1.3

24.2 ± 3.1

>30

>30

1e

H

13.9 ± 1.7

10.0 ± 1.2

6.7 ± 0.9

4.1 ± 1.6

18.9 ± 6.6

17.4 ± 3.6

11.8 ± 2.6

1f

Ac

24.7 ± 3.3

7.1 ± 1.2

>30

19.8 ± 2.5

24.2 ± 3.9

29.1 ± 6.6

>30

1g

H

11.4 ± 1.2

13.5 ± 0.4

11.5 ± 0.7

14.8 ± 1.3

n.d.

17.8 ± 1.6

8.8 ± 0.6

1h

Ac

15.0 ± 3.2

2.7 ± 0.4

>30

14.1 ± 1.7

>30

16.0 ± 2.1

>30

1i

H

>30

>30

>30

>30

n.d.

>30

>30

1j

Ac

>30

10.4 ± 1.0

>30

>30

>30

>30

>30

OA

H

61.3 ± 10.0

58.2 ± 3.7

44.9 ± 6.2

49.1 ± 3.5

64.1 ± 8.6

66.7 ± 4.6

57.9 ± 9.3

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22.5 ± 3.3

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1b

In general, the compounds (with R1 = H) show only moderate cytotoxic activity. The

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substituent R2 seems to exert no relevant influence on the cytotoxicity of the compounds [except for compound 1i whose EC50 value was significantly higher for all tumor cell lines (EC50 > 30)]. As far as the selectivity (tumor/non-tumor cells) is concerned, the significance

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of the substitution at position C-3 was particularly apparent. Acetylation led to a remarkable increase of the malignant/nonmalignant selectivity. Nonmalignant NIH 3T3 cells were less affected by acetylated compounds, and a significant increase in the cytotoxicity for the

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ovarian carcinoma cell line A2780 was observed. Selectivity [characterized by the selectivity index (SI; e.g. SI = EC50 (NIH 3T3)/EC50 (A2780)] is - with respect to prospective pharmacological applications - of great interest, and compound 1h has to be highlighted in this context. The combination of an acetoxy moiety at position C-3 and a benzyl group as R2 led to the highest selectivity index (SI > 11) in this series of compounds. Furthermore, this compound was also characterized by the highest cytotoxicity for A2780 cells (EC50 = 2.7 ± 0.4 µM). Encouraged by these results further Ugi products were synthetized starting with variations of the isocyanide component. First, the 4-UCR were performed with different aliphatic isocyanides (n-butyl isocyanide, iso-propyl-isocyanide while the amine component remained 5

ACCEPTED MANUSCRIPT unchanged for better comparability of the biological results. We refrained from synthesizing other furfuryl derivatives holding different substituents due to a reduced selectivity of these compounds between malignant and nonmalignant cells. The syntheses of the OA-Ugi derivatives were completed by a direct acetylation of the crude products. This facilitated the purification of the obtained Ugi products. OA-Ugi derivatives holding a hydroxyl group at

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position C-3 were synthesized by a deacetylation with potassium hydroxide. The results of the

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biological screening of products 2a, 2b and 3a-3f is given in Table 2.

Scheme 2. Synthesis of OA-Ugi products 2a, 2b and 3a-3f: a. MeOH, rt, 7–12 days, then b. Ac2O, pyridine, rt, 12h, 46–78%; c. KOH, MeOH, 65 °C, 6h, 87–98%. Table 2. Cytotoxicity of compounds 2a, 2b and 3a-3f; EC50 values in µM from SRB assay after 96 h of treatment; the values are averaged from three independent experiments performed each in triplicate; confidence interval CI = 95%; n.d. not detected/not determined. Human cancer cell lines: 518A2 (melanoma), A2780 (ovarian carcinoma), HT29 (colorectal 6

ACCEPTED MANUSCRIPT carcinoma), MCF7 (breast carcinoma), A549 (lung adenocarcinoma), FaDu (hypopharyngeal carcinoma with multidrug resistance) and nonmalignant mouse fibroblasts (NIH 3T3).

EC50

R1

2a

R2

A2780

HT29

MCF7

A549

FaDu

NIH 3T3

Ac

6.5 ± 0.3

3.6 ± 0.4

9.6 ± 1.1

16.7 ± 1.8

7.5 ± 2.0

12.5 ± 4.8

10.1 ± 1.6

2b

H

5.5 ± 2.4

5.9 ± 2.6

4.9 ± 1.8

4.9 ± 0.2

3a

Ac

15.0 ± 2.0

3.1 ± 0.3

>30

>30

3b

H

>30

19.3 ± 1.2

>30

21.3 ± 3.7

3c

Ac

11.3 ± 0.9

5.6 ± 0.5

>30

13.9 ± 4.6

3d

H

>30

17.6 ± 0.6

15.9 ± 1.7

3e

Ac

>30

3.0 ± 0.4

3f

H

>30

>30

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518A2

7.9 ± 0.4

5.5 ± 2.3

>30

16.1 ± 1.1

>30

n.d.

>30

>30

16.2 ± 1.6

17.9 ± 3.2

>30

15.5 ± 1.5

n.d.

>30

>30

>30

22.1 ± 4.6

>30

22.7 ± 0.1

>30

>30

>30

n.d.

>30

>30

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n.d.

7

ACCEPTED MANUSCRIPT Scheme 3. Synthesis of OA-Ugi products 4a-4j: a. MeOH, rt, 7–12 days, then b. Ac2O, pyridine, rt, 12h, 64–89%; c. KOH, MeOH, 65 °C, 6h, 82–99%.

The variation of the isocyanide component was continued by using benzyl isocyanide. The obtained Ugi products consisted of a terminal benzyl amide. This structural feature is a

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predominant element in different promising studies concerning the biological activities of triterpenoids [26, 28, 33-35].

The good EC50 values of this type of Ugi products are compiled in Table 3. The cytotoxicity of compound 4a-4j essentially concerned A2780 cells line, while cytotoxic effect for other

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cell lines was significantly lower. Thus, for the nonmalignant cell line NIH 3T3 EC50 values greater 30 µM were determined (except for compound 4c).

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Table 3. Cytotoxicity of compounds 4a-4j; EC50 values in µM from SRB assay after 96 h of treatment; the values are averaged from three independent experiments performed each in triplicate; confidence interval CI = 95%; n.d. not detected/not determined. Human cancer cell lines: 518A2 (melanoma), A2780 (ovarian carcinoma), HT29 (colorectal carcinoma), MCF7 (breast carcinoma), A549 (lung adenocarcinoma), FaDu (hypopharyngeal carcinoma with

EC50 R1

R2

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multidrug resistance) and nonmalignant mouse fibroblasts (NIH 3T3).

518A2

A2780

HT29

MCF7

A549

FaDu

NIH 3T3

Ac

8.2 ± 0.4

4.4 ± 0.2

>30

>30

>30

19.8± 0.1

>30

4b

H

>30

>30

15.6 ± 7.6

24.6 ± 1.9

n.d.

>30

>30

4c

Ac

20.9 ± 2.5

8.3 ± 0.5

14.1 ± 1.0

9.7 ± 0.7

14.9 ± 1.2

10.1 ± 0.9

10.2 ± 1.8

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4a

H

>30

15.4 ± 2.1

>30

19.4± 1.6

n.d.

>30

>30

Ac

18.9 ± 1.9

6.0 ± 0.2

>30

19.2 ± 0.7

n.d.

15.5 ± 1.5

>30

H

>30

12.9 ± 2.4

>30

>30

>30

>30

>30

4g

Ac

15.9 ± 1.2

6.3 ± 0.1

>30

15.0 ± 1.6

>30

22.1 ± 10.2

>30

4h

H

>30

13.2 ± 2.4

>30

12.6 ± 2.5

>30

>30

>30

4i

Ac

13.5 ± 1.4

5.4 ± 0.4

>30

>30

>30

20.3 ± 3.1

>30

4d

4e

4f

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H

>30

>30

>30

>30

n.d.

>30

>30

Using methyl isocyanoacetate (Scheme 4), gave Ugi products holding a dipeptide structure as

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exemplified in compounds 5a and 5b.

Scheme 4. Synthesis of OA-Ugi products 5a and 5b: a. MeOH, rt, 7–12 days, then b. Ac2O,

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pyridine, rt, 12h, 46–51%.

This variation of the structure resulted in a significant decrease of the tumor/nontumor cell selectivity (SI = 2.5, respectively SI = 2.1). Furthermore, compound 5a and 5b (Tab. 4)

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showed - compared to the acetylated OA-Ugi products containing a terminal amide structure a markedly higher cytotoxicity for the nonmalignant NIH 3T3 cells (EC50 = 8.9 ± 1.2 µM and

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EC50 = 7.3 ± 0.6 µM, respectively). Again, a variation of the R2 moiety resulted in negligible small effects on the EC50 values. Table 4. Cytotoxicity of compounds 5a and 5b; EC50 values in µM from SRB assay after 96 h of treatment; the values are averaged from three independent experiments performed each in triplicate; confidence interval CI = 95%. Human cancer cell lines: 518A2 (melanoma), A2780 (ovarian carcinoma), HT29 (colorectal carcinoma), MCF7 (breast carcinoma), A549 (lung adenocarcinoma), FaDu (hypopharyngeal carcinoma with multidrug resistance) and nonmalignant mouse fibroblasts (NIH 3T3).

EC50

R1

R2

518A2

A2780

HT29

MCF7

A549

8505C

NIH 3T3

9

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Ac

7.7 ± 0.3

3.5 ± 0.5

11.1 ± 0.5

4.6 ± 0.4

5b

Ac

6.5 ± 0.1

3.5 ± 0.4

14.3 ± 0.7

7.0 ± 0.9

6.3 ± 0.8

6.5 ± 0.7

12.2 ± 0.5

7.3 ± 0.6

5.9 ± 0.2

8.9 ± 1.2

The final series of Ugi products of this investigation was characterized by a variation of the

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acid component. In general, previous and extensive studies showed derivatives of maslinic acid (MA) - compared to their OA analogs and depending on the number of acetoxy moieties attached to positions C-2 and C-3 [36] - to possess significantly increased cytotoxicity. Thus, four selected Ugi products of MA were synthesized to evaluate their pharmacological

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potential as well. Due to their better solubility, the derivatives were synthesized in one step directly from 2,3-di-O-acetyl-MA (Ac-MA) (Scheme 5). Parent Ac-MA was synthesized

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from OA according to literature [37].

Scheme 5. Synthesis of MA-Ugi products 6a, 6b, 7a and 7b: a. MeOH, rt, 7–12 days, 38– 64%.

Again, an increased cytotoxicity (compared to OA analogs) was experienced for these MAUgi products. EC50 values in the low µM range were determined (Table 5). In this study, compound 7b showed the highest cytotoxicity (EC50 = 0.3 ± 0.1 µM) for ovarian carcinoma cells A2780, thus being twenty-five times more cytotoxic than the corresponding OAderivative (EC50 = 5.4 ± 0.4 µM) and 65 times more cytotoxic than parent MA. Moreover, 7b 10

ACCEPTED MANUSCRIPT is characterized by a remarkable tumor/non-tumor cell selectivity (SI = 73.0). A graphical comparison of the EC50 values and the selectivity indices of the OA- and the MA-derivatives is depicted (Diagram 2 in the Supplementary Material). Table 5. Cytotoxicity of compounds 6a, 6b, 7a, 7b and maslinic acid (MA, for comparison)

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and acetylated maslinic acid (Ac-MA, for comparison); EC50 values in µM from SRB assay after 96 h of treatment; the values are averaged from three independent experiments performed each in triplicate; confidence interval CI = 95%; n.d. not detected/not determined. Human cancer cell lines: 518A2 (melanoma), A2780 (ovarian carcinoma), HT29 (colorectal carcinoma), MCF7 (breast carcinoma), A549 (lung adenocarcinoma), FaDu (hypopharyngeal

EC50

R1

6a

R2

A2780

HT29

Ac

2.9 ± 0.2

0.5 ± 0.1

3.9 ± 0.4

6b

Ac

3.9 ± 0.4

0.4 ± 0.3

7a

Ac

13.1 ± 5.2

7b

Ac

Ac-MA MA

MCF7

A549

8505C

NIH 3T3

4.1 ± 1.2

4.4 ± 0.7

4.8 ± 0.8

7.8 ± 0.5

3.4 ± 0.4

4.0 ± 0.6

3.5 ± 0.3

3.0 ± 0.3

7.3 ± 1.8

2.1 ± 6.0

9.9 ± 5.5

3.4 ± 1.9

5.4 ± 3.9

1.2 ± 0.2

0.3 ± 0.1

2.1 ± 0.3

2.1 ± 0.5

2.2 ± 0.2

2.1 ± 0.3

21.9 ± 7.1

Ac

n.d.

10.5 ± 0.9

25.5 ± 2.2

1.6 ± 1.5

18.9 ± 1.5

21.3 ± 1.3

12.2 ± 0.8

H

13.7 ± 0.9

19.5 ± 0.8

28.8 ± 0.5

37.2 ± 1.2

23.4 ± 0.5

17.0 ± 1.0

21.1 ± 0.2

14.1 ± 10.2

6.5 ± 1.7

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carcinoma with multidrug resistance) and nonmalignant mouse fibroblasts (NIH 3T3).

Considering the high pharmacological potential and the remarkable selectivity index,

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compound 7b was subjected to further investigations concerning its mode of action. Thus, ovarian carcinoma cells A2780 were treated for 48h with 7b and for comparison with 6a. Two different concentration (1.2 µM or 2 µM) of 7b were applied for the analysis of concentrationdependent changes in the morphology of the cells. Fluorescence microscopic examinations of the cells in an AO/PI (acridine orange/propidium iodide) dye exclusion test showed distinct morphological changes (see Supplementary Material). The dead cells showed intact cell membranes, indicating a non-necrotic, controlled cell death mechanism. In addition, annexin V-FITC/PI staining assays were performed to determine the apoptosis-inducing activity. Cells treated with compound 7b (2 µM) showed an increase of annexin V-positive cells; the same was true for 6a. These results revealed also that cell death is not exclusively triggered by apoptosis. Finally, the influence of the compounds onto the cell cycle was analyzed by a flow11

ACCEPTED MANUSCRIPT cytometric DNA distribution assay (for detailed information see Supplementary Material). Thereby, indications for an apoptotic cell death were observed by the appearance of a subG1phase. To sum up, the results indicated that the compound 6a and 7b induced programmed cell death in part through the pathway of apoptosis.

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Furthermore, three Passerini products 8-10 were synthesized and the cytotoxicity (Table 6) of these α-acyloxy carboxamides was compared to that of the products from the Ugi reactions. The P-3CR comprises the reaction of an isocyanide, a carbonyl component and a carboxylic acid (Scheme 6). Compared to the U-4CR an increased reaction rate was observed,

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presumably caused by an increased solubility of the acidic component in DCM.

Scheme 6. Synthesis of Passerini products 8-10: a. DCM, rt, 24h, 58–90%.

Table 6. Cytotoxicity of compounds 8-10: EC50 values in µM from SRB assay after 96 h of treatment; the values are averaged from three independent experiments performed each in triplicate; confidence interval CI = 95%. Human cancer cell lines: 518A2 (melanoma), A2780 (ovarian carcinoma), HT29 (colorectal carcinoma), MCF7 (breast carcinoma), A549 (lung adenocarcinoma), 8505C (thyroid carcinoma) and nonmalignant mouse fibroblasts (NIH 3T3). 12

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518A2

A2780

HT29

MCF7

A549

8505C

NIH 3T3

8

H

13.2 ± 2.4

8.8 ± 0.8

27.4 ± 4.4

12.9 ± 4.3

13.2 ± 3.9

12.7 ± 1.7

20.0 ± 6.3

9

H

7.7 ± 0.3

6.8 ± 0.1

11.3 ± 0.3

6.4 ± 0.5

6.9 ± 0.3

7.2 ± 0.1

6.4 ± 0.8

10

OAc

4.3 ±0.5

1.0 ± 0.1

4.9 ± 0.8

3.6 ± 1.2

3.2 ± 1.0

7.3 ± 1.0

2.0 ± 0.4

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EC50

Concluding, in the biological evaluation good EC50 values were measured for the Passerini products; the values were comparable to those measured for the investigated Ugi products of

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Ac-OA and Ac-MA. A distinct decrease of the selectivity index, however, was observed thus

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reducing the pharmacological potential of this α-acyloxy carboxamides.

3. Conclusion

In summary, a library of 37 triterpenoid derivatives was synthesized by U-4CRs or P-3CRs and SRB assays allowed to determine their cytotoxicity. In general, the compounds showed moderate to good cytotoxicity for several human tumor cell lines. Especially, MA-Ugi products 6a, 7a and 7b gave remarkable EC50 values in the low µM range and a high

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tumor/non-tumor cell selectivity. Furthermore, several structure-activity relationships have been identified: Thus, varying the substrate showed acetylated products and compounds holding a terminal benzylamide moiety to be higher selective, while the presence of ester groups initiated a loss of this selectivity. Further biological assays showed that treating A2780

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cells with compounds 6a or 7b led to a non-necrotic, controlled cell death in part occurring

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through the pathway of apoptosis.

4. Experimental part

4.1 General Chemistry

Reagents were bought from commercial suppliers and used without any further purification. The solvents were dried according to usual procedures. TLC was performed on silica gel (Macherey–Nagel, detection with ceriummolybdate spray reagent). Melting points are uncorrected (LEICA hot stage microscope). Microanalysis were performed with a Foss– Heraeus Vario EL (CHNS) instrument. NMR spectra were recorded using the VARIAN spectrometers Gemini 2000 or Unity 500 at 27 °C (δ given in ppm; J in Hz, typical experiments: DQF–COSY, HMBC, HSQC). ESI–MS spectra were taken on a Finnigan MAT LCQ 7000 (electrospray, voltage 4.1 kV, sheath gas nitrogen) instrument. The optical 13

ACCEPTED MANUSCRIPT rotations were measured on a Perkin–Elmer polarimeter 341 at 20 °C; IR spectra were recorded on a Perkin–Elmer FT–IR spectrometer Spectrum 1000; UV–vis spectra were recorded on Perkin–Elmer Lambda 14 spectrometer.

4.2 General procedures (GP)

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4.2.1 GP A: Ugi–multicomponent reaction of triterpenoic acids Paraformaldehyde (1 equiv.) was suspended in dry methanol (0.05 mL/mg triterpenoic acid), followed by the addition of amine (1.2 equiv.). The suspension was stirred for two hours at room temperature. The triterpenoic acid (1 equiv.) and the corresponding isocyanide (1

mixture was poured into aqueous HCl (2

M)

phase was washed with aqueous NaOH (2

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equiv.) were added, and the solution was stirred for additional 7 to 12 days. The reaction and was extracted with DCM (3x). The organic

M,

3x), brine and dried with sodium sulfate. The

chromatographic purification.

4.2.2 GP B: Acetylation

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solvent was removed under diminished pressure, and the residue was subjected to

The starting material (1 equiv.) was dissolved in dry pyridine, followed by the addition of

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acetic anhydride (2 equiv.) in presence of catalytic amounts of DMAP. After 12 h of stirring at room temperature the mixture was poured into an ice-cold solution of aqueous HCl (5%), the precipitate was filtered off and purified by chromatography.

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4.2.3 GP C: Deacetylation

Potassium hydroxide (10 equiv.) was dissolved in MeOH (0.20 mL/mg triterpenoic acid) and acetylated triterpenoic acid derivative (1 equiv.) was added. After 6h of stirring at 65 °C the

AC C

solution was neutralized with aqueous HCl (2

M)

and extracted with DCM (3x). The organic

phase was washed with brine and dried with sodium sulfate. The solvent was removed under diminished pressure, and the residue was subjected to chromatography.

4.2.4 GP D: Passerini–multicomponent reaction of triterpenoic acids The acetylated triterpenoic acid (1 equiv.) was dissolved in DCM (0.02 mL/mg triterpenoic acid), paraformaldehyde (1 equiv.) and isocyanide (1 equiv.) were added; stirring at room temperature was continued for another 24 h. The reaction mixture was diluted with DCM and washed with HCl (aq., 2

M,

3x) and brine. The organic phase was dried with sodium sulfate,

14

ACCEPTED MANUSCRIPT and the solvent was removed under diminished pressure. The residue was subjected to chromatography.

4.3 Syntheses 4.3.1

(3β)

3-Hydroxy-N-(2-(tert-butylamino)-2-oxoethyl)-N-methyl-olean-12-en-28-

RI PT

carboxamide (1a) Compound 1a was prepared according to GP A by reaction of OA (200 mg, 0.44 mmol) with methylamine (40 % in H2O, 50 µL), tert–butyl isocyanide (50 µL, 0.44 mmol) and paraformaldehyde (14 mg, 0.47 mmol). After column chromatography (silica gel, ethyl

SC

acetate/chloroform, 1:9) 1a (150 mg, 59 %) was obtained as a colorless solid; RF = 0.44 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 132–134 °C; [α]D = –27.28° (c = 0.63, CHCl3); IR (KBr): v = 3428s, 2946s, 2872s, 1670s, 1616s, 1544m, 1456s,

M AN U

1388s, 1364s, 1270m, 1224m, 1202m, 1104m, 1030m, 998m cm–1; 1H NMR (400 MHz, CDCl3) δ = 6.28 (s, 1H, NH), 5.26 (dd, J = 3.5, 3.5 Hz, 1H, H–12), 4.22 (br s, 1H, H–32a), 3.39 (br s, 1H, H–32b), 3.22–3.18 (m, 1H, H–3), 3.18 (s, 3H, H–31), 3.12 (dd, J = 14.1, 2.7 Hz, 1H, H–18), 2.11 (ddd, J = 14.3, 14.3, 3.2 Hz, 1H, H–16a), 1.97–1.74 (m, 3H, H–11a + H– 11b + H–16b), 1.74–1.48 (m, 7H, H–19a + H–1a + H–22a + H–22b + H–15a + H–6a + H–9),

TE D

1.47–1.30 (m, 3H, H–21a + H–7a + H–6b), 1.29 (s, 9H, H–35), 1.28–1.21 (m, 3H, H–21b + H–7b + H–19b), 1.13 (s, 3H, H–27), 1.12–1.05 (m, 1H, H–15b), 0.97 (s, 3H, H–23), 0.93 (s, 3H, H–30), 0.97–0.91 (m, 1H, H–1b), 0.90 (s, 3H, H–29), 0.88 (s, 3H, H–25), 0.77 (s, 3H, H– 24), 0.74–0.70 (m, 1H, H–5), 0.69 (s, 3H, H–26) ppm;

13

C NMR (100 MHz, CDCl3): δ =

EP

177.8 (C28), 169.2 (C33), 144.7 (C13), 122.0 (C12), 79.1 (C3), 56.3 (C32), 55.4 (C5), 51.1 (C34), 47.9 (C9), 47.8 (C17), 46.4 (C19), 43.2 (C18), 42.1 (C14), 39.3 (C8), 38.9 (C4), 38.5 (C1), 37.8 (C31), 37.2 (C10), 34.0 (C21), 33.1 (C29), 32.9 (C7), 30.6 (C20), 29.8 (C22), 28.9

AC C

(C35), 28.2 (C23), 27.9 (C15), 27.3 (C2), 26.2 (C27), 24.1 (C30), 23.5 (C11), 22.4 (C16), 18.4 (C6), 17.1 (C26), 15.7 (C24), 15.4 (C25) ppm; MS (ESI): m/z (%) = 583.3 ([M+H]+, 98), 605.5 ([M+Na]+, 5), 1165.4 ([2M+H]+, 100), 1187.6 ([2M+Na]+, 25); analysis calculated for C37H62N2O3 (582.90): C 76.24, H 10.72, N 4.81; found: C 76.01, H 10.94, N 4.57. 4.3.2

(3β)

3-Acetoxy-N-(2-(tert-butylamino)-2-oxoethyl)-N-methyl-olean-12-en-28-

carboxamide (1b) Compound 1b was prepared by acetylation according to GP B from 1a (54 mg, 0.09 mmol) followed by column chromatography (silica gel, n-hexane/ethyl acetate, 7:3), and 1b (50 mg, 86 %) was obtained as a colorless solid; RF = 0.65 (silica gel, toluene/ethyl acetate/n– 15

ACCEPTED MANUSCRIPT heptane/formic acid, 80:30:10:3); mp = 136–138 °C; [α]D = –28.62° (c = 0.31, CHCl3); IR (KBr): v = 3440s, 2948vs, 2876m, 1734s, 1682s, 1670s, 1630s, 1618s, 1542m, 1466m, 1456m, 1390m, 1366s, 1246vs, 1204w, 1182w, 1102w, 1074w, 1028m, 1006m cm–1; 1H NMR (500 MHz, CDCl3): δ = 6.27 (s, 1H, NH), 5.26 (dd, J = 3.1, 3.1 Hz, 1H, H–12), 4.50–4.45 (m, 1H, H–3), 4.23 (br s, 1H, H–33a), 3.38 (br s, 1H, H–33b), 3.18 (s, 3H, H–31), 3.12 (dd, J =

RI PT

2.9, 2.9 Hz, 1H, H–18), 2.11 (ddd, J = 13.1, 13.1, 2.9 Hz, 1H, H–16a), 2.03 (s, 3H, H–37), 1.97–1.84 (m, 2H, H–11a + H–11b), 1.82–1.75 (m, 1H, H–16b), 1.74–1.65 (m, 2H, H–19a + H–22a), 1.66–1.47 (m, 7H, H–9, H–1a + H–22b + H–15a + H–2a + H–2b + H–6a), 1.48–1.31 (m, 3H, H–7a + H–21a + H–6b), 1.29 (s, 9H, H–35), 1.28–1.16 (m, 3H, H–21b + H–7b + H–

SC

19b), 1.13 (s, 3H, H–27), 1.12–1.05 (m, 1H, H–15b), 1.05–0.97 (m, 1H, H–1b), 0.93 (s, 3H, H–30), 0.91 (s, 3H, H–25), 0.91 (s, 3H, H–29), 0.85 (s, 3H, H–23), 0.84 (s, 3H, H–24), 0.83– 0.80 (m, 1H, H–5), 0.69 (s, 3H, H–26) ppm; 13C NMR (125 MHz, CDCl3): δ = 177.7 (C28),

M AN U

171.1 (C36), 169.1 (C33), 144.7 (C13), 121.9 (C12), 81.1 (C3), 56.3 (C32), 55.5 (C5), 51.0 (C34), 47.8 (C9), 47.8 (C17), 46.4 (C19), 43.2 (C18), 42.1 (C14), 39.3 (C8), 38.2 (C1), 37.8 (C4), 37.8 (C31), 37.1 (C10), 34.0 (C21), 33.1 (C29), 32.8 (C7), 30.6 (C20), 29.8 (C22), 28.9 (C35), 28.2 (C23), 27.9 (C15), 26.2 (C27), 24.1 (C30), 23.7 (C2), 23.5 (C11), 22.4 (C16), 21.4 (C37), 18.3 (C6), 17.1 (C26), 16.8 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 625.3

TE D

([M+H]+, 100), 647.6 ([M+Na]+, 25), 679.3 ([M+Na+MeOH]+, 10), 1271.5 ([2M+Na]+, 40); analysis calculated for C39H64N2O4 (624.94): C 74.95, H 10.32, N 4.48; found: C 74.69, H 10.51, N 4.27.

(3β) 3-Hydroxy-N-(2-(tert-butylamino)-2-oxoethyl)-N-(prop-2-enyl)-olean-12-en-28-

carboxamide (1c)

EP

4.3.3

Compound 1c was prepared according to GP A by reaction of OA (200 mg, 0.44 mmol) with

AC C

allylamine (40 µL, 0.53 mmol), tert–butyl isocyanide (50 µL, 0.44 mmol) and paraformaldehyde (14 mg, 0.47 mmol). Column chromatography (silica gel, ethyl acetate/chloroform, 1:9) afforded 1c (170 mg, 64 %) as a colorless solid; RF = 0.54 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 123–125 °C; [α]D = –27.24° (c = 0.38, CHCl3); IR (KBr): v = 3432s, 3334s, 2948s, 2872s, 1670s, 1616s, 1544s, 1454s, 1390s, 1364s, 1224s, 1206s, 1182s, 1048m, 1002s, 926m, 756s cm–1; 1H NMR (400 MHz, CDCl3) δ = 6.17 (br s, 1H, NH), 5.75 (ddd, J = 22.1, 10.9, 5.8 Hz, 1H, H–32), 5.30–5.18 (m, 3H, H–12 + H–33a + H–33b), 4.31 (d, J = 11.5 Hz, 1H, H–31a), 4.20 (d, J = 14.9 Hz, 1H, H– 34a), 3.95 (d, J = 10.8 Hz, 1H, H–31b), 3.44 (br s, 1H, H–34b), 3.19 (dd, J = 11.0, 4.5 Hz, 1H, H–3), 3.08 (dd, J = 14.9, 3.7 Hz, 1H, H–18), 2.11 (ddd, J = 14.1, 14.1, 2.5 Hz, 1H, H– 16

ACCEPTED MANUSCRIPT 16a), 2.00–1.79 (m, 2H, H–11a + H–11b), 1.77–1.62 (m, 4H, H–19a + H–22a + H–22b + H– 16b), 1.62–1.48 (m, 6H, H–6a + H–15a + H–2a + H–2b + H–1a + H–9), 1.47–1.33 (m, 3H, H–6b + H–7a + H–21a), 1.30 (s, 9H, H–37), 1.28–1.14 (m, 3H, H–7b + H–21b + H–19b), 1.13 (s, 3H, H–27), 1.10–1.02 (m, 1H, H–15b), 0.97 (s, 3H, H–23), 0.97–0.94 (m, 1H, H–1b), 0.92 (s, 3H, H–30), 0.89 (s, 6H, H–29 + H–25), 0.77 (s, 3H, H–24), 0.72 (s, 3H, H–26), 0.72– 13

C NMR (100 MHz, CDCl3) δ = 177.4 (C28), 168.9 (C35), 144.7

RI PT

0.69 (m, 1H, H–5) ppm;

(C13), 132.5 (C32), 122.0 (C12), 119.1 (C33), 79.1 (C3), 55.4 (C5), 52.3 (C34), 52.0 (C31), 51.1 (C36), 48.0 (C17), 47.9 (C9) 46.8 (C19), 43.5 (C18), 42.0 (C14), 39.3 (C8) , 38.9 (C4), 38.5 (C1), 37.2 (C10), 34.2 (C21), 33.0 (C29), 33.0 (C7), 30.5 (C22), 30.3 (C20), 28.9 (C37), 28.4 (C15), 28.2 (C23), 27.3 (C2), 26.0 (C27), 24.1 (C30), 23.5 (C11), 22.8 (C16), 18.5 (C6),

SC

17.2 (C26), 15.7 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 609.3 ([M+H]+, 28), 615.6 ([M+Li]+, 8), 631.5 ([M+Na]+, 10), 647.4 ([M+K]+, 6), 1224.7 ([2M+Li]+, 20), 1239.5

M AN U

([2M+Na]+, 100), 1256.3 ([2M+K]+, 10); analysis calculated for C39H64N2O3 (608.94): C 76.92, H 10.59, N 4.60; found: C 76.84, H 10.73, N 4.35.

4.3.4

(3β)

3-Acetoxy-N-(2-(tert-butylamino)-2-oxoethyl)-N-(prop-2-enyl)-olean-12-en-28-

carboxamide (1d)

TE D

Compound 1d was prepared according to GP B by acetylation of 1c (130 mg, 0.21 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 8:2) afforded 1d (132 mg, 95 %) as a colorless solid; RF = 0.76 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 116–118 °C; [α]D = –30.72° (c = 0.38, CHCl3); IR (KBr): v = 3342m,

EP

2950s, 2876s, 1734s, 1682s, 1616s, 1540m, 1454s, 1392s, 1365s, 1246s, 1206m, 1182m, 1028m, 1006m, 988m, 924m cm–1; 1H NMR (500 MHz, CDCl3) δ = 6.17 (br s, 1H, NH), 5.75 (ddd, J = 22.3, 11.0, 5.8 Hz, 1H, H–32), 5.29–5.19 (m, 3H, H–12 + H–33a + H–33b), 4.50–

AC C

4.45 (m, 1H, H–3), 4.33 (br s, 1H, H–31a), 4.20 (d, J = 13.3 Hz, 1H, H–34a), 3.95 (d, J = 12.1 Hz, 1H, H–31b), 3.43 (br s, 1H, H–34b), 3.08 (dd, J = 14.9, 3.0 Hz, 1H, H–18), 2.11 (ddd, J = 14.2, 14.2, 2.8 Hz, 1H, H–16a), 2.03 (s, 3H, H–39), 1.93 (ddd, J = 18.2, 11.2, 3.3 Hz, 1H, H– 11a), 1.85 (ddd, J = 18.7, 6.5, 4.0 Hz, H–11b) 1.77–1.65 (m, 3H, H–22a + H–16b + H–19a), 1.64–1.47 (m, 7H, H–22b + H–2a + H–2b + H–6a + H–1a + H–9 + H–15a), 1.47–1.31 (m, 3H, H–7a + H–6b + H–21a), 1.29 (s, 9H, H–37), 1.26–1.15 (m, 3H, H–7b + H–21b + H–19b), 1.12 (s, 3H, H–27), 1.09–0.99 (m, 2H, H–1b + H–15b), 0.92 (s, 3H, H–30), 0.91 (s, 3H, H– 29), 0.89 (s, 3H, H–25), 0.85 (s, 3H, H–23), 0.84 (s, 3H, H–24), 0.84–0.80 (m, 1H, H–5), 0.72 (s, 3H, H–26) ppm; 13C NMR (125 MHz, CDCl3) δ = 177.5 (C28), 171.1 (C38), 168.9 (C35), 144.7 (C13), 132.5 (C32), 121.9 (C12), 119.1 (C33), 81.1 (C3), 55.5 (C5), 52.3 (C34), 52.0 17

ACCEPTED MANUSCRIPT (C31), 51.1 (C36), 48.0 (C17), 47.8 (C9), 46.7 (C19), 43.5 (C18), 42.0 (C14), 39.3 (C8), 38.2 (C1), 37.8 (C4), 37.1 (C10), 34.2 (C21), 33.0 (C29), 32.9 (C7), 30.5 (C22), 30.3 (C20), 28.9 (C37), 28.4 (C15), 28.2 (C23), 25.9 (C27), 24.1 (C30), 23.7 (C2), 23.5 (C11), 22.8 (C16), 21.4 (C39), 18.3 (C6), 17.2 (C26), 16.8 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 651.4 ([M+H]+, 100), 673.6 ([M+Na]+, 20), 683.3 ([M+H+MeOH]+, 10), 995.9 ([3M+K+H]2+, 10),

C 75.65, H 10.22, N 4.30; found: C 75.40, H 10.33, N 4.13.

RI PT

1301.3 ([2M+H]+, 50), 1324.6 ([2M+Na]+, 60); analysis calculated for C41H66N2O4 (650.97):

4.3.5 (3β) 3-Hydroxy-N-(2-(tert-butylamino)-2-oxoethyl)-N-(furan-2-ylmethyl)-olean-12-en-

SC

28-carboxamide (1e)

Compound 1e was prepared according to GP A by reaction of OA (200 mg, 0.44 mmol) with furfurylamine (47 µL, 0.51 mmol), tert–butyl isocyanide (50 µL, 0.44 mmol) and

M AN U

paraformaldehyde (14 mg, 0.47 mmol). Column chromatography (silica gel, ethyl acetate/chloroform, 1:9) gave 1e (227 mg, 80 %) as a colorless solid; RF = 0.57 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 128–130 °C; [α]D = –2.30° (c = 0.63, CHCl3); IR (KBr): ν = 3434br, 2946s, 1667s, 1622m, 1540m, 1454m, 1390m, 1365m, 1260m, 1224m, 1178m, 1076m, 1032m, 1012m, 755m cm–1; 1H NMR (400 MHz, CDCl3) δ =

TE D

7.34 (dd, J = 1.8, 0.8 Hz, 1H, H–33), 6.32 (dd, J = 3.2, 1.9 Hz, 1H, H–34), 6.26 (m, 1H, H– 35), 5.96 (s, 1H, NH), 5.25 (dd, J = 3.3, 3.3 Hz, 1H, H–12), 4.76 (d, J = 15.6 Hz, 1H, H–31a), 4.63 (d, J = 15.9 Hz, 1H, H–31b), 4.19 (d, J = 15.9 Hz, 1H, H–36a), 3.60 (d, J = 15.2 Hz, 1H, H–36b), 3.19 (dd, J = 11.1, 4.9 Hz, 1H, H–3), 3.09 (dd, J = 11.2, 3.2 Hz, 1H, H–18), 2.13

EP

(ddd, J = 14.3, 14.3, 2.8 Hz, 1H, H–16a), 1.98–1.84 (m, 2H, H–11a + H–11b), 1.84–1.74 (m, 2H, H–16b + H–22a), 1.74–1.63 (m, 3H, H–19a + H–22b + H–15a), 1.63–1.47 (m, 5H, H–9 + H–1a + H–2a + H–2b + H–6a), 1.46–1.29 (m, 3H, H–6b + H–7a + H–21a), 1.27 (s, 9H, H–

AC C

39), 1.25–1.14 (m, 3H, H–19b + H–21b + H–7b), 1.12 (s, 3H, H–27), 1.04 (ddd, J = 14.2, 2.9, 2.9 Hz, 1H, H–15b), 0.97 (s, 3H, H–23), 0.96–0.93 (m, 1H, H–1b), 0.92 (s, 3H, H–30), 0.89 (s, 3H, H–25), 0.88 (s, 3H, H–29), 0.76 (s, 3H, H–24), 0.74–0.69 (m, 1H, H–5), 0.69 (s, 3H, H–26) ppm; 13C NMR (100 MHz, CDCl3) δ = 177.4 (C28), 168.4 (C37), 150.2 (C32), 144.7 (C13), 142.4 (C33), 121.9 (C12), 110.7 (C34), 109.3 (C35), 79.1 (C3), 55.4 (C5), 52.8 (C36), 51.1 (C38), 48.3 (C17), 47.9 (C9), 46.8 (C19), 46.3 (C31), 43.6 (C18), 42.0 (C14), 39.3 (C8), 38.9 (C4), 38.5 (C1), 37.2 (C10), 34.2 (C21), 33.0 (C29), 33.0 (C7), 30.5 (C22), 30.5 (C20), 28.8 (C39), 28.4 (C15), 28.2 (C23), 27.3 (C2), 25.9 (C27), 24.1 (C30), 23.5 (C11), 23.1 (C16), 18.5 (C6), 17.2 (C26), 15.7 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 649.3 ([M+H]+, 100), 671.5 ([M+Na]+, 12), 993.1 ([3M+K+H]2+, 4), 1298.5 ([2M+H]+, 12), 1320.5 18

ACCEPTED MANUSCRIPT ([2M+Na]+, 94); analysis calculated for C41H88N2O4 (648.96): C 75.88, H 9.94, N 4.32; found: C 75.69, H 10.09, N 4.17.

4.3.6 (3β) 3-Acetoxy-N-(2-(tert-butylamino)-2-oxoethyl)-N-(furan-2-ylmethyl)-olean-12-en28-carboxamide (1f)

RI PT

Compound 1f (100 mg, 0.15 mmol) was prepared according to GP B by acetylation of 1e. Column chromatography (silica gel, n-hexane/ethyl acetate, 8:2) gave 1f (93 mg, 88 %) as a colorless solid; RF = 0.39 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 111–112 °C; [α]D = –2.43° (c = 0.66, CHCl3); IR (KBr): v = 3422s, 2948s, 1734s,

SC

1684s, 1638s, 1540m, 1454s, 1392s, 1366s, 1246s, 1178m, 1162m, 1028s, 1010s cm–1; 1H NMR (500 MHz, CDCl3) δ = 7.35 (dd, J = 1.1, 0.9 Hz, 1H, H–33), 6.33 (dd, J = 3.2, 1.9 Hz, 1H, H–34), 6.27 (dd, J = 3.2, 0.9 Hz, 1H, H–35), 5.97 (s, 1H, NH), 5.26 (dd, J = 3.2, 3.2 Hz,

M AN U

1H, H–12), 4.77 (d, J = 15.1 Hz, 1H, H–31a), 4.64 (d, J = 15.8 Hz, 1H, H–31b), 4.51–4.46 (m, 1H, H–3), 4.20 (d, J = 15.9 Hz, 1H, H–36a), 3.69–3.48 (br s, 1H, H–36b), 3.10 (dd, J = 13.6, 3.7 Hz, 1H, H–18), 2.14 (ddd, J = 14.2, 14.2, 2.8 Hz, 1H, H–16a), 2.03 (s, 3H, H–41), 1.97–1.74 (m, 4H, H–11a + H–11b + H–16b + H–22a), 1.70 (m, 2H, H–19a + H–22b), 1.65– 1.49 (m, 5H, H–1a + H–15a + H–2a+ H–2b + H–9), 1.45 (m, 1H, H–6a), 1.42–1.33 (m, 3H,

TE D

H–6b + H–21a + H–7a), 1.27 (s, 9H, H–39), 1.27–1.14 (m, 3H, H–21b + H–7b + H–19b), 1.12 (s, 3H, H–27), 1.09–0.98 (m, 2H, H–1b + H–15b), 0.93 (s, 3H, H–30), 0.92 (s, 3H, H– 25), 0.90 (s, 3H, H–29), 0.85 (s, 3H, H–23), 0.85 (s, 3H, H–24), 0.84–0.80 (m, 1H, H–5), 0.70 (s, 3H, H–26) ppm; 13C NMR (125 MHz, CDCl3) δ = 177.5 (C28), 171.2 (C40), 168.5 (C37),

EP

150.2 (C32),144.8 (C13), 142.5 (C33), 121.8 (C12), 110.7 (C34), 109.4 (C35), 81.1 (3), 55.5 (C5), 52.8 (C36), 51.2 (C38), 48.3 (C17), 47.8 (C9), 46.8 (C19), 46.3 (C31), 43.6 (C18), 42.1 (C14), 39.3 (C8), 38.2 (C1), 37.8 (C4), 37.1 (C10), 34.2 (C21), 33.0 (C29), 32.9 (C7), 30.5

AC C

(C22), 30.5 (C20), 28.8 (C39), 28.4 (C15), 28.2 (C23), 25.9 (C27), 24.1 (C30), 23.7 (C2), 23.5 (C11), 23.1 (C16), 21.4 (C41), 18.4 (C6), 17.2 (C26), 16.8 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 691.3 ([M+H]+, 100), 713.6 ([M+Na]+, 10), 1056.0 ([3M+K+H]2+, 5), 1381.0 ([2M+H]+ 5), 1403.4 ([2M+Na]+, 45); analysis calculated for C43H66N2O5 (691.00): C 74.74, H 9.63, N 4.05; found: C 74.47, H 9.80, N 3.79.

4.3.7

(3β)

3-Hydroxy-N-(2-(tert-butylamino)-2-oxoethyl)-N-benzyl-olean-12-en-28-

carboxamide (1g) Compound 1g was prepared according to GP A by reaction of OA (200 mg, 0.44 mmol) with benzylamine (60 µL, 0.55 mmol), tert–butyl isocyanide (50 µL, 0.44 mmol) and 19

ACCEPTED MANUSCRIPT paraformaldehyde (14 mg, 0.47 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 7:3) gave 1g (171 mg, 59 %) as a colorless solid; RF = 0.54 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 141–144 °C; [α]D = +12.73° (c = 0.33, CHCl3); IR (KBr): v = 3430s, 3332m, 2946vs, 2870s, 1668s, 1616s, 1542m, 1496m, 1454s, 1390s, 1364s, 1300w, 1284m, 1266m, 1224m, 1204m, 1182m, 1048m, 1030m, 1002m, 732m

RI PT

cm–1; 1H NMR (500 MHz, CDCl3): δ = 7.35–7.31 (m, 2H, H–34), 7.29–7.25 (m, 1H, H–35), 7.20 (d, J = 7.3 Hz, 2H, H–33), 5.90 (s, 1H, NH), 5.32–5.12 (br s, 1H, H–12), 4.94 (d, J = 15.2 Hz, 1H, H–31a), 4.76 (br s, 1H, H–31b), 4.17–3.99 (br s, 1H, H–36a), 3.64 (br s, 1H, H– 36b), 3.20 (dd, J = 11.3, 4.5 Hz, 1H, H–3), 3.14–3.01 (m, 1H, H–18), 2.09 (ddd, J = 14.6,

SC

13.8, 2.7 Hz, 1H, H–16a), 2.00–1.81 (m, 2H, H–11a + H–11b), 1.80–1.64 (m, 5H, H–22a + H–22b + H–16a + H–15a + H–19a), 1.65–1.48 (m, 5H, H–1a + H–2a + H–2b + H–6a + H–9), 1.47–1.36 (m, 2H, H–7a + H–6b), 1.36–1.30 (m, 1H, H–21a) 1.28 (s, 9H, H–39), 1.26–1.13

M AN U

(m, 3H, H–7b + H–21b + H–19b), 1.12 (s, 3H, H–27),1.03–0.99 (m, 1H, H–15b), 0.98 (s, 3H, H–23), 0.98–0.93 (m, 1H, H–1b), 0.92 (s, 3H, H–30), 0.91 (s, 3H, H–25), 0.89 (s, 3H, H–29), 0.80 (s, 3H, H–26), 0.78 (s, 3H, H–24), 0.75–0.70 (m, 1H, H–5) ppm; 13C NMR (125 MHz, CDCl3): δ = 177.9 (C28), 168.5 (C37), 144.8 (C13), 136.8 (C32), 128.8 (C34), 127.6 (C35), 127.4 (C33), 121.9 (C12), 79.2 (C3), 55.5 (C5), 52.9 (C31), 52.6 (C36), 51.2 (C38), 48.3

TE D

(C17), 47.9 (C9), 47.0 (C19), 43.5 (C18), 42.1 (C14), 39.4 (C8), 38.9 (C1), 38.6 (C10), 37.3 (C4), 34.3 (C21), 33.1 (C7), 33.0 (C29), 30.5 (C20), 30.5 (C22), 28.9 (C39), 28.5 (C15), 28.3 (C23), 27.4 (C2), 25.9 (C27), 24.2 (C30), 23.5 (C11), 23.3 (C16), 18.5 (C6), 17.5 (C26), 15.7 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 659.3 ([M+H]+, 100), 681.5 ([M+Na]+, 10),

EP

1008.1 ([3M+K+H]2+, 5), 1318.3 ([2M+H]+, 15), 1340. 5 ([2M+Na]+, 50); analysis calculated for C43H66N2O3 (658.99): C 78.37, H 10.09, N 4.25; found: C 78.07, H 10.24, N 4.02. (3β)

3-Acetoxy-N-(2-(tert-butylamino)-2-oxoethyl)-N-benzyl-olean-12-en-28-

AC C

4.3.8

carboxamide (1h)

Compound 1h was prepared by acetylation according to GP B of 1g (88 mg, 0.13 mmol) followed by column chromatography (silica gel, n-hexane/ethyl acetate, 8:2) to afford 1h (87 mg, 93 %) as a colorless solid; RF = 0.76 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 130–131 °C; [α]D = +12.56° (c = 0.36, CHCl3); IR (KBr): v = 3428s, 2948vs, 2876m, 1734s, 1682s, 1636s, 1538m, 1454m, 1392s, 1364s, 1246s, 1180m, 1028m, 1006m, 986m cm–1; 1H NMR (500 MHz, CDCl3) δ = 7.35–7.29 (m, 2H, H–34), 7.28–7.24 (m, 1H, H–35), 7.22–7.17 (m, 2H, H–33), 5.90 (s, 1H, NH), 5.19 (m, 1H, H–12), 4.93 (d, J = 15.5 Hz, 1H, H–31a), 4.82–4.68 (br s, 1H, H–31b), 4.51–4.45 (dd, J = 8.0, 7.9 Hz, 1H, H–3), 20

ACCEPTED MANUSCRIPT 4.19–3.93 (br s, 1H, H–36a), 3.76–3.51 (br s, 1H, H–36b), 3.06 (m, 1H, H–18), 2.15–2.05 (ddd, J = 14.7, 14.7, 2.9 Hz, 1H, H–16a), 2.03 (s, 3H, H–41), 1.98–1.88 (m, 1H, H–11a), 1.88–1.80 (ddd, J = 18.6, 6.0, 4.0 Hz, 1H, H–11b), 1.80–1.64 (m, 5H, H–19a + H–15a + H– 16b + H–22a + H–22b), 1.64–1.58 (m, 3H, H–1a + H–2a + H–2b), 1.58–1.49 (m, 2H, H–9 + H–6a), 1.47–1.30 (m, 3H, H–6b + H–21a + H–7a), 1.27 (s, 9H, H–39), 1.25–1.12 (m, 2H, H–

RI PT

21b + H–7b), 1.11 (s, 3H, H–27), 1.08–0.95 (m, 2H, H–15b + H–1b), 0.93 (s, 3H, H–25), 0.92 (s, 3H, H–30), 0.88 (s, 3H, H–29), 0.85 (s, 6H, H–23 + H–24), 0.86–0.80 (m, 1H, H–5), 0.79 (s, 3H, H–26) ppm; 13C NMR (125 MHz, CDCl3) δ = 177.9 (C28), 171.1 (C40), 168.4 (C37), 144.8 (C13), 136.7 (C32), 128.8 (C34), 127.6 (C35), 127.4 (C33), 121.8 (C12), 81.1 (C3),

SC

55.5 (C5), 52.9 (C31), 52.5 (C36), 51.2 (C38), 48.3 (C9), 47.8 (C19), 46.9 (C17), 43.5 (C18), 42.0 (C14), 39.4 (C8), 38.2 (C1), 37.8 (C10), 37.1 (C4), 34.2 (C21), 33.0 (C29), 33.0 (C7), 30.5 (C20), 30.5 (C22), 28.8 (C39), 28.4 (C15), 28.2 (C23), 25.9 (C27), 24.2 (C30), 23.6

M AN U

(C2), 23.5 (C11), 23.3 (C16), 21.4 (C41), 18.4 (C6), 17.5 (C26), 16.8 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 701.4 ([M+H]+, 100), 723.6 ([M+Na]+, 10), 735.3 ([M+H+MeOH]+, 4), 1071.2 ([3M+K+H]2+, 4), 1401.0 ([2M+H]+, 10), 1423.6 ([2M+Na]+, 30); analysis calculated for C45H68N2O4 (701.03): C 77.10, H 9.78, N 4.00; found: C 76.82, H 9.96, N 3.86.

carboxamide (1i)

TE D

4.3.9 (3β) 3-Hydroxy-N-(2-(tert-butylamino)-2-oxoethyl)-N-(3-fluorobenzyl)-olean-12-en-28-

Compound 1i was prepared according to GP A by reaction of OA (250 mg, 0.55 mmol) with 3-fluorobenzylamine (75 µL, 0.66 mmol), tert-butyl isocyanide (62 µL, 0.55 mmol) and

EP

paraformaldehyde (20 mg, 0.67 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 7:3) gave 1i (183 mg, 61 %) as a colorless solid; RF = 0.57 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 145–147 °C; [α]D = –17.63° (c = 0.32,

AC C

CHCl3); IR (KBr): v = 3442vs, 3432vs, 2946vs, 2870m, 1668s, 1616s, 1592m, 1542m, 1486m, 1454s, 1390m, 1364m, 1284w, 1268m, 1252m, 1224m, 1180m, 1048w, 1004m, 732m, 684m cm–1; 1H NMR (400 MHz, CDCl3): δ = 7.30 (ddd, J = 7.9, 7.9, 6.0 Hz, 1H, H–Ph)), 7.02–6.88 (m, 3H, H–Ph), 5.89 (br s, 1H, NH), 5.17 (br s, 1H, H–12), 4.93 (d, J = 15.9 Hz, 1H, H–31a), 4.77 (br s, 1H, H–31b), 4.04 (br s, 1H, H–38a), 3.66 (br s, 1H, H–38b), 3.20 (dd, J = 11.1, 4.8 Hz, 1H, H–3)), 3.03 (dd, J = 10.7, 2.0 Hz, 1H, H–18), 2.10 (ddd, J = 14.5, 14.5, 1.9 Hz, 1H, H–16a), 2.00–1.79 (m, 2H, H–11a + H–11b), 1.79–1.64 (m, 5H, H–16b + H–22a + H–22b + H–19a + H–15a), 1.64–1.49 (m, 5H, H–1a + H–9 + H–2a + H–2b + H–6a), 1.47–1.32 (m, 3H, H–6b + H–7a + H–21a), 1.29 (s, 9H, H–41), 1.27–1.13 (m, 3H, H–21b + H–7b + H–19b), 1.12 (s, 3H, H–27), 1.06–0.98 (m, 1H, H–15b), 0.98 (s, 3H, H–23), 0.97–0.93 (m, 1H, H–1b), 21

ACCEPTED MANUSCRIPT 0.92 (s, 3H, H–30), 0.91 (s, 3H, H–25), 0.89 (s, 3H, H–29), 0.79 (s, 3H, H–26), 0.78 (s, 3H, H–24), 0.74–0.70 (m, 1H, H–5) ppm;

13

C NMR (100 MHz, CDCl3): δ = 178.0 (C28), 168.2

(C39), 163.2 (d, JCF = 246.7 Hz, Ph), 144.7 (C13), 139.5 (d, JCF = 6.5 Hz, Ph), 130.39 (d, JCF = 8.2 Hz, Ph), 122.57 (d, J = 95.7 Hz, Ph), 122.1 (C12), 114.6 (d, JCF = 21.1 Hz, Ph), 114.6 (d, JCF = 21.1 Hz, Ph), 79.2 (C3), 55.4 (C5), 52.6 (C31), 52.5 (C38), 51.3 (C40), 48.3 (C17),

RI PT

47.9 (C9), 47.0 (C19), 43.5 (C18), 42.1 (C14), 39.4 (C8), 38.9 (C4), 38.5 (C1), 37.3 (C10), 34.2 (C21), 33.0 (C7), 33.0 (C29), 30.6 (C20), 30.5 (C22), 28.9 (C41), 28.5 (C15), 28.3 (C23), 27.4 (C2), 26.0 (C27), 24.2 (C30), 23.5 (C11), 23.4 (C16), 18.5 (C6), 17.6 (C26), 15.7 (C24), 15.5 (C25) ppm;

19

F NMR (375 MHz, CDCl3) δ = –112.38 (s) ppm; MS (ESI): m/z

(%) = 677.3 ([M+H]+, 65), 691.3 [M+H-H2O+MeOH]+, 20), 699.5 ([M+Na]+, 10), 1353.2

M AN U

76.29, H 9.68, N 4.14; found: C 75.94, H 9.71, N 3.87.

SC

([2M+H]+, 5), 1375.6 ([2M+Na]+, 100); analysis calculated for C43H65FN2O3 (676.98): C

4.3.10 (3β) 3-Acetoxy-N-(2-(tert-butylamino)-2-oxoethyl)-N-(3-fluorobenzyl)-olean-12-en-28carboxamide (1j)

Compound 1j was prepared according to GP B by acetylation of 1i (93 mg, 0.14 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 7:3) afforded 1j (90 mg, 95 %) as

TE D

a colorless solid; RF = 0.75 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 125–127 °C; [α]D = –17.63° (c = 0.37, CHCl3); IR (KBr): v = 3426m, 3346m, 2949vs, 2876m, 1734s, 1682s, 1682m, 1617s, 1592m, 1539m, 1453s, 1392s, 1365s, 1248vs, 1180m, 1027m, 1005m cm–1; 1H NMR (400 MHz, CDCl3): δ = 7.30 (ddd, J = 7.9,

EP

7.9, 5.9 Hz, 1H, H–Ph), 7.01–6.87 (m, 3H, H–Ph), 5.88 (br s, 1H, NH), 5.17 (br s, 1H, H–12), 4.93 (d, J = 16.2 Hz, 1H, H–31a), 4.77 (br s, 1H, H–31b), 4.48 4.48 (dd, J = 8.3, 7.8 Hz, 1H, H–3), 4.12–3.95 (br s, 1H, H–38a), 3.66 (br s, 1H, H–38b), 3.03 (dd, J =8.0, 1.9 Hz, 1H, H–

AC C

18), 2.11 (ddd, J = 15.2, 15.2, 3.0 Hz, 1H, H–16a), 2.04 (s, 3H, H–43), 1.99–1.80 (m, 2H, H– 11a + H–11b), 1.78–1.66 (m, 5H, H–22a + H–22b + H–19a + H–16b + H–15a), 1.69–1.55 (m, 3H, H–1a + H–2a + H–2b), 1.57–1.46 (m, 2H, H–6a + H–9), 1.45–1.38 (m, 1H, H–7a), 1.39– 1.30 (m, 2H, H–6b + H–21a), 1.29 (s, 9H, H–41), 1.28–1.22 (m, 2H, H–21b + H–7b), 1.22– 1.12 (m, 1H, H–19b), 1.11 (s, 3H, H–27), 1.10–0.98 (m, 2H, H–1b + H–15b), 0.94 (s, 3H, H– 25), 0.92 (s, 3H, H–30), 0.89 (s, 3H, H–29), 0.86 (s, 6H, H–23 + H–24), 0.85–0.81 (m, 1H, H–5), 0.79 (s, 3H, H–26) ppm; 13C NMR (100 MHz, CDCl3): δ = 178.0 (C28), 171.1 (C42), 168.2 (C39), 163.2 (d, JCF = 246.7 Hz, Ph), 144.7 (C13), 139.5 (d, JCF = 4.3 Hz, Ph), 130.4 (d, JCF = 8.2 Hz, Ph), 123.1 (d, J = 3.8 Hz, Ph), 122.0 (C12), 114.6 (d, JCF = 21.1 Hz, Ph), 114.5 (d, JCF = 21.1 Hz, Ph), 81.1 (C3), 55.5 (C5), 52.6 (C31), 52.4 (C38), 51.3 (C40), 48.3 (C17), 22

ACCEPTED MANUSCRIPT 47.8 (C9), 46.9 (C19), 43.4 (C18), 42.0 (C14), 39.4 (C1), 38.2 (C8), 37.8 (C4), 37.1 (C10), 34.2 (C21), 33.0 (C29), 32.9 (C7), 30.6 (C20), 30.5 (C22), 28.9 (C41), 28.4 (C15), 28.2 (C23), 25.9 (C27), 24.2 (C30), 23.7 (C2), 23.5 (C11), 23.4 (C16), 21.4 (C43), 18.4 (C6), 17.5 (C26), 16.8 (C24), 15.5 (C25) ppm;

19

F NMR (375 MHz, CDCl3) δ = –112.38 (s) ppm; MS

(ESI): m/z (%) = 719.3 ([M+H]+, 100), 741.7 ([M+Na]+, 10), 779.3 ([M+K]+, 8), 1090.2

RI PT

([3M+H+Na]2+, 5), 1438.1 ([2M+H]+, 5), 1460.6 ([2M+Na]+, 50), 1508.9 ([2M+K]+, 48); analysis calculated for C45H67FN2O4 (719.02): C 75.17, H 9.39, N 3.90; found: C 74.82, H 9.51, N 3.75.

SC

4.3.11 (3β) 3-Acetoxy-N-(2-(1-methylethyl)-2-oxoethyl)-N-(furan-2-ylmethyl)-olean-12-en-28carboxamide (2a)

Compound 2a was prepared according to GP A by reaction of OA (250 mg, 0.55 mmol) with

M AN U

furfurylamine (50 µL, 0.68 mmol), iso–propyl isocyanide (50 µL, 0.53 mmol) and paraformaldehyde (20 g, 0.67 mmol). Reaction according to GP B and column chromatography (silica gel, n-hexane/ethyl acetate, 7:3) yielded 2a (290 mg, 78 %) as a colorless solid; RF = 0.54 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 117–119 °C; [α]D = +5.74° (c = 0.39, CHCl3); IR (KBr): v = 3424m, 3336m, 2947vs, 2876m, 1734s, 1680s, 1638s, 1624s, 1539m, 1465m, 1366s, 1247vs, 1176s, 1147m, 1076w,

TE D

1027m, 1008m, 1027m, 986m, 733m cm–1; 1H NMR (400 MHz, CDCl3): δ = 7.35 (dd, J = 1.6, 0.7 Hz, 1H, H–33), 6.32 (dd, J = 3.2, 1.8 Hz, 1H, H–34), 6.27 (d, J = 3.1 Hz, 1H, H–35), 6.00 (d, J = 6.7 Hz, 1H, NH), 5.26 (dd, J = 3.0, 3.0 Hz, 1H, H–12), 4.80 (d, J = 15.7 Hz, 1H, H–

EP

31a), 4.66 (d, J = 15.9 Hz, 1H, H–31b), 4.53–4.43 (m, 1H, H–3), 4.19 (d, J = 15.7 Hz, 1H, H– 36a), 3.96 (m, 1H, H–38), 3.67 (d, J = 13.5 Hz, 1H, H–36b), 3.08 (d, J = 11.5 Hz, 1H, H–18), 2.14 (ddd, J = 14.2, 14.2, 3.2 Hz, 1H, H–16a), 2.03 (s, 3H, H–42), 1.97–1.84 (m, 2H, H–11a +

AC C

H–11b), 1.85–1.65 (m, 4H, H–16b + H–22a + H–22b + H–19a), 1.66–1.48 (m, 5H, H–9 + H– 1a + H–15a + H–2a + H–2b), 1.48–1.31 (m, 3H, H–6b + H–21a + H–7a), 1.26–1.15 (m, 3H, H–19b + H–21b + H–7b), 1.12 (s, 3H, H–27), 1.09 (d, J = 6.6 Hz, 3H, H–39), 1.07 (d, J = 6.6 Hz, 3H, H–40), 1.06–0.97 (m, 2H, H–1b + H–15b), 0.92 (s, 3H, H–30), 0.91 (s, 3H, H–25), 0.90 (s, 3H, H–29), 0.85 (s, 3H, H–23), 0.84 (s, 3H, H–24), 0.83–0.79 (m, 1H, H–5), 0.70 (s, 3H, H–26) ppm;

13

C NMR (100 MHz, CDCl3): δ = 177.5 (C28), 171.1 (C40), 168.4 (C37),

150.1 (C32), 144.8 (C13), 142.5 (C33), 121.8 (C12), 110.6 (C34), 109.3 (C35), 81.1 (C3), 55.5 (C5), 52.1 (C36), 48.3 (C17), 47.8 (C9), 46.7 (C19), 46.4 (C31), 43.6 (C18), 42.0 (C14), 41.4 (C38), 39.3 (C8), 38.2 (C1), 37.8 (C4), 37.1 (C10), 34.2 (C21), 33.0 (C29), 32.9 (C7), 30.5 (C20), 30.5 (C22), 28.3 (C15), 28.2 (C23), 25.9 (C27), 24.1 (C30), 23.7 (C2), 23.5 23

ACCEPTED MANUSCRIPT (C11), 23.1 (C16), 22.8 (C39), 22.7 (C40), 21.4 (C42), 18.4 (C6), 17.2 (C26), 16.8 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 677.3 ([M+H]+, 100), 699.5 ([M+Na]+, 20), 1035.1 ([3M+K+H]2+, 5), 1353.1 ([2M+H]+, 80), 1375.5 ([2M+Na]+, 96); analysis calculated for C42H64N2O5 (676.97): C 74.52, H 9.53, N 4.14; found: C 74.36, H 9.81, N 4.02.

RI PT

4.3.12 (3β) 3-Hydroxy-N-(2-(1-methylethyl)-2-oxoethyl)-N-(furan-2-ylmethyl)-olean-12-en28-carboxamide (2b)

Compound 2b was prepared according to GP C by deacetylation of 2a (100 mg, 0.15 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 7:3) gave 2b (91 mg, 98 %) as

SC

colorless solid; RF = 0.43 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 133–136 °C; [α]D = +4.61° (c = 0.32, CHCl3); IR (KBr): v = 3442vs, 3426vs, 2944vs, 2874m, 1716w, 1662s, 1624s, 1542m, 1508w, 1466m, 1386m, 1366m, 1250w, 1224w, 1200w,

M AN U

1176m, 1146w, 1076w, 1048w, 1030m, 1012m, 734m cm–1; 1H NMR (500 MHz, CDCl3): δ = 7.39–7.32 (m, 1H, H–33), 6.32 (dd, J = 3.1, 1.9 Hz, 1H, H–34), 6.27 (d, J = 3.1 Hz, 1H, H– 35), 6.02 (d, J = 7.2 Hz, 1H, NH), 5.26 (dd, J = 3.3, 3.3 Hz, 1H, H–12), 4.80 (d, J = 15.0 Hz, 1H, H–31a), 4.66 (d, J = 15.9 Hz, 1H, H–31b), 4.19 (d, J = 15.7 Hz, 1H, H–36a), 3.96 (dddd, J = 13.4, 6.7, 6.7, 6.7 Hz, 1H, H–38), 3.66 (d, J = 11.6 Hz, 1H, H–36b), 3.20 (dd, J = 11.4,

TE D

4.5 Hz, 1H, H–3), 3.08 (dd, J = 11.9, 3.2 Hz, 1H, H–18), 2.14 (ddd, J = 14.3, 2.7 Hz, 1H, H– 16a), 1.97–1.83 (m, 2H, H–11a + H–11b), 1.83–1.66 (m, 4H, H–16b + H–1a + H–2b + H– 19a), 1.66–1.48 (m, 2H, H–9 + H–6a), 1.47–1.40 (m, 1H, H–7a), 1.40–1.31 (m, 2H, H–21a + H–6b), 1.25–1.15 (m, 3H, H–19b + H–21b + H–7b), 1.12 (s, 3H, H–27), 1.09 (d, J = 6.6 Hz,

EP

3H, H–39), 1.07 (d, J = 6.6 Hz, 3H, H–40), 1.06–0.99 (m, 1H, H–15b), 0.97 (s, 3H, H–23), 0.97–0.93 (m, 1H, H–1b), 0.92 (s, 3H, H–30), 0.89 (s, 3H, H–29), 0.89 (s, 3H, H–25), 0.77 (s, 3H, H–24), 0.74–0.70 (m, 1H, H–5), 0.70 (s, 3H, H–26) ppm; 13C NMR (125 MHz, CDCl3): δ

AC C

= 177.5 (C28), 168.4 (C37), 150.1 (C32), 144.8 (C13), 142.5 (C33), 121.9 (C12), 110.6 (C34), 109.3 (C35), 79.1 (C3), 55.4 (C5), 52.1 (C36), 48.3 (C17), 47.9 (C9), 46.8 (C19), 46.4 (C31), 43.6 (C18), 42.1 (C14), 41.4 (C38), 39.3 (C8), 38.9 (C4), 38.5 (C1), 37.2 (C10), 34.2 (C21), 33.0 (C29), 33.0 (C7), 30.5 (C22), 30.5 (C20), 28.3 (C15), 28.2 (C23), 27.3 (C2), 26.0 (C27), 24.1 (C30), 23.5 (C11), 23.1 (C16), 22.8 (C39), 22.7 (C40), 18.5 (C6), 17.2 (C26), 15.7 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 635.3 ([M+H]+, 20), 657.6 ([M+Na]+, 10), 1269.4 ([2M+H]+, 8), 1291.5 ([2M+Na]+, 100); analysis calculated for C40H62N2O4 (634.93): C 75.67, H 9.84, N 4.41; found: C 75.46, H 10.07, N 4.11.

24

ACCEPTED MANUSCRIPT 4.3.13

(3β)

3-Acetoxy-N-(2-butylamino-2-oxoethyl)-N-(prop-2-enyl)-olean-12-en-28-

carboxamide (3a) Compound 3a was prepared according to GP A by reaction of OA (250 mg, 0.55 mmol) with allylamine (49 µL, 0.65 mmol), n–butyl isocyanide (57 µL, 0.58 mmol) and paraformaldehyde (16 mg, 0.53 mmol). Acetylation according to GP B and column

RI PT

chromatography (silica gel, n-hexane/ethyl acetate, 8:2) gave 3a (200 g, 59 %) as a colorless solid; RF = 0.57 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 95–97 °C; [α]D = +20.27° (c = 0.39, CHCl3); IR (KBr): v = 3432m, 3342m, 2948vs, 2874m, 1734s, 1684s, 1666m, 1640s, 1618s, 1542m, 1466s, 1390m, 1378m, 1246vs, 1206m, 1182m,

SC

1108w, 1028m, 1006m, 988m cm–1; 1H NMR (500 MHz, CDCl3): δ = 6.45 (s, 1H, NH), 5.75 (ddd, J = 16.7, 10.9, 5.8 Hz, 1H, H–32), 5.31–5.18 (m, 3H, H–12 + H–33a + H–33b), 4.51– 4.44 (m, 1H, H–3), 4.36 (br s, 1H, H–31a), 4.14 (d, J = 14.7 Hz, 1H, H–34a), 4.02 (dd, J =

M AN U

15.5, 5.1 Hz, 1H, H–31b), 3.58 (br s, 1H, H–34b), 3.25 (dddd, J = 13.5, 6.9, 6.9, 6.9 Hz, 1H, H–36a), 3.12 (dddd, J = 12.1, 6.3, 6.3, 6.3 Hz, 1H, H–36b), 3.05 (d, J = 12.1 Hz, 1H, H–18), 2.12 (ddd, J = 14.2, 14.2, 2.9 Hz, 1H, H–16a), 2.03 (s, 3H, H–41), 1.96–1.82 (m, 2H, H–11a + H–11b), 1.75–1.63 (m, 4H, H–16b + H–19a + H–22a + H–22b), 1.63–1.54 (m, 5H, H–9 + H– 1a + H–15a + H–2a + H–2b), 1.54–1.48 (m, 1H, H–6a), 1.48–1.34 (m, 5H, H–6b + H–7a +

TE D

H–37a + H–37b + H–21a), 1.34–1.28 (m, 2H, H–38a + H–38b), 1.28–1.14 (m, 3H, H–21b + H–7b + H–19b), 1.13 (s, 3H, H–27), 1.09–0.99 (m, 2H, H–1b + H–15b), 0.92 (s, 6H, H–30 + H–25), 0.90 (s, 3H, H–29), 0.89 (t, J = 7.3 Hz, 3H, H–39), 0.86 (s, 3H, H–23), 0.84 (s, 3H, H–24), 0.84–0.81 (m, 1H, H–5), 0.72 (s, 3H, H–26) ppm; 13C NMR (125 MHz, CDCl3): δ =

EP

177.7 (C28), 171.1 (C40), 169.7 (C35), 144.8 (C13), 132.4 (C32), 121.8 (C12), 119.3 (C33), 81.1 (C3), 55.5 (C5), 52.2 (C31), 51.6 (C34), 48.0 (C9), 47.8 (C17), 46.7 (C19), 43.6 (C18), 42.0 (C14), 39.3 (C8), 39.1 (C36), 38.2 (C1), 37.8 (C4), 37.1 (C10), 34.2 (C21), 33.1 (C29),

AC C

32.9 (C7), 31.7 (C37), 30.5 (C22), 30.4 (C20), 28.3 (C15), 28.2 (C23), 26.0 (C27), 24.1 (C30), 23.7 (C2), 23.5 (C11), 22.8 (C16), 21.4 (C41), 20.2 (C38), 18.3 (C6), 17.2 (C26), 16.8 (C24), 15.5 (C25), 13.9 (C39) ppm; MS (ESI): m/z (%) = 651.4 ([M+H]+, 100), 673.5 ([M+Na]+, 10), 705.5 ([M+K]+, 10) 988.0 ([3M+Na+H]2+, 10), 1301.5 ([2M+H]+, 10), 1323.5 ([2M+Na]+, 80), 1339.5 ([2M+K]+, 75); analysis calculated for C41H66N2O4 (650.97): C 75.65, H 10.22, N 4.30; found: C 75.43, H 10.37, N 4.03.

4.3.14

(3β)

3-Hydroxy-N-(2-butylamino-2-oxoethyl)-N-(prop-2-enyl)-olean-12-en-28-

carboxamide (3b)

25

ACCEPTED MANUSCRIPT Compound 3b was prepared according to GP C by deacetylation of 3a (87 mg, 0.13 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 6:4) gave 3b (76 mg, 98 %) as a colorless solid; RF = 0.49 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 123–124 °C; [α]D = –14.85° (c = 0.32, CHCl3); IR (KBr): v = 3442vs, 2948vs, 2872s, 1668s, 1616s, 1548m, 1464m, 1400m, 1386m, 1364m, 1300w, 1282w, 1250w, 1224w, 1206m,

RI PT

1182m, 1048m, 1032m, 1002m, 732m cm–1; 1H NMR (400 MHz, CDCl3): δ = 6.46 (br s, 1H, NH), 5.75 (ddd, J = 16.5, 11.2, 5.7 Hz, 1H, H–32), 5.33–5.17 (m, 3H, H–12 + H–33a + H– 33b), 4.36 (d, J = 13.8 Hz, 1H, H–31a), 4.13 (d, J = 14.8 Hz, 1H, H–34a), 4.02 (dd, J = 16.4, 5.7 Hz, 1H, H–31b), 3.58 (d, J = 10.6 Hz, 1H, H–34b), 3.30–3.17 (m, 2H, H–36a + H–3),

SC

3.13 (ddd, J = 13.1, 6.4, 6.4 Hz, 1H, H–36b), 3.05 (dd, J = 12.4, 12.4 Hz, 1H, H–18), 2.12 (ddd, J = 14.3, 11.6, 2.6 Hz, 1H, H–16a), 1.98–1.80 (m, 2H, H–11a + H–11b), 1.78–1.63 (m, 4H, H–16b + H–22a + H–22b + H–19a), 1.64–1.49 (m, 6H, H–15a + H–2a + H–2b + H–1a +

M AN U

H–9 + H–6a), 1.48–1.36 (m, 4H, H–6b + H–7a + H–37a + H–37b), 1.36–1.25 (m, 3H, H–21a + H–38a + H–38b), 1.25–1.15 (m, 3H, H–7b + H–21b + H–1b), 1.13 (s, 3H, H–27), 1.06 (ddd, J = 13.6, 3.5, 2.7 Hz, 1H, H–15b), 0.98 (s, 3H, H–23), 0.92 (s, 3H, H–30), 0.89 (s, 6H, H–25 + H–29), 0.89 (t, J = 7.3 Hz, 3H, H–39), 0.77 (s, 3H, H–24), 0.76–0.73 (m, 1H, H–5), 0.72 (s, 3H, H–16) ppm; 13C NMR (100 MHz, CDCl3): δ = 177.7 (C28), 169.7 (C35), 144.8

TE D

(C13), 132.4 (C32), 121.9 (C12), 119.3 (C33), 79.1 (C3), 55.4 (C5), 52.2 (C31), 51.6 (C34), 48.0 (C9), 47.9 (C17), 46.7 (C19), 43.6 (C18), 42.0 (C14), 39.3 (C36), 39.1 (C8), 38.9 (C4), 38.5 (C1), 37.2 (C10), 34.2 (C21), 33.1 (C29), 32.9 (C7), 31.7 (C37), 30.5 (C22), 30.3 (C20), 28.3 (C15), 28.2 (C23), 27.3 (C2), 26.0 (C27), 24.1 (C30), 23.5 (C11), 22.8 (C16), 20.2

EP

(C38), 18.4 (C6), 17.2 (C26), 15.7 (C24), 15.5 (C25), 13.9 (C39) ppm; MS (ESI): m/z (%) = 609.3 ([M+H]+, 30), 631.5 ([M+Na]+, 10), 1217.3 ([2M+H]+, 8), 1239.6 ([2M+Na]+, 100); analysis calculated for C39H64N2O3 (608.93): C 76.92, H 10.59, N 4.60; found: C 76.77, H

AC C

10.71, N 4.43.

4.3.15

(3β)

3-Acetoxy-N-(2-butylamino-2-oxoethyl)-N-(furan-2-ylmethyl)-olean-12-en-28-

carboxamide (3c)

Compound 3c was prepared according to GP A by reaction of OA (250 mg, 0.55 mmol) with furfurylamine (58 µL, 0.66 mmol), n–butyl isocyanide (57 µL, 0.58 mmol) and paraformaldehyde (16 mg, 0.53 mmol). Reaction according to GP B and column chromatography (silica gel, n-hexane/ethyl acetate, 8:2) gave 3c (173 mg, 46 %) as a colorless solid; RF = 0.64 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 115–117 °C; [α]D = +4.43° (c = 0.37, CHCl3); IR (KBr): v = 3428m, 2948vs, 2874m, 1734s, 26

ACCEPTED MANUSCRIPT 1684m, 1638s, 1540m, 1506w, 1466m, 1390m, 1372m, 1246vs, 1200w, 1178m, 1148m, 1076w, 1028m, 1010m cm–1; 1H NMR (500 MHz, CDCl3): δ = 7.35 (dd, J = 1.6, 0.7 Hz, 1H, H–33), 6.33 (dd, J = 3.0, 1.8 Hz, 1H, H–34), 6.28 (d, J = 3.1 Hz, 1H, H–35), 6.22 (br s, 1H, NH), 5.26 (dd, J = 3.1, 3.1 Hz, 1H, H–12), 4.82 (d, J = 15.7 Hz, 1H, H–31a), 4.69 (d, J = 15.9 Hz, 1H, H–31b), 4.51–4.46 (m, 1H, H–3), 4.15 (d, J = 15.4 Hz, 1H, H–36a), 3.70 (br s, 1H,

RI PT

H–36b), 3.19 (dddd, J = 13.4, 6.9, 6.9, 6.9 Hz, 1H, H–38a), 3.15–3.09 (m, 1H, H–38b), 3.09– 3.04 (m, 1H, H–18), 2.15 (ddd, J = 14.9, 14.3, 2.9 Hz, 1H, H–16a), 2.03 (s, 3H, H–43), 1.96– 1.85 (m, 2H, H–11a + H–11b), 1.85–1.77 (m, 2H, H–16b + H–22a), 1.76–1.67 (m, 2H, H–22b + H–19a), 1.65–1.54 (m, 5H, H–2a + H–2b + H–15a + H–1a + H–9), 1.54–1.49 (m, 1H, H–

SC

6a), 1.47–1.33 (m, 5H, H–6b + H–7a + H–21a + H–39a + H–39b), 1.34–1.27 (m, 2H, H–40a + H–40b), 1.27–1.17 (m, 3H, H–7b + H–21b + H–19b), 1.13 (s, 3H, H–27), 1.08–0.99 (m, 2H, H–1b + H–15b), 0.93 (s, 3H, H–30), 0.92 (s, 3H, H–25), 0.90 (s, 3H, H–29), 0.89 (t, J =

3H, H–26) ppm;

13

M AN U

7.2 Hz, 3H, H–41), 0.85 (s, 3H, H–23), 0.85 (s, 3H, H–24), 0.84–0.81 (m, 1H, H–5), 0.70 (s, C NMR (125 MHz, CDCl3): δ = 177.7 (C28), 171.1 (C42), 169.3 (C37),

150.0 (C32), 144.9 (C13), 142.6 (C33), 121.8 (C12), 110.6 (C34), 109.4 (C35), 81.1 (C3), 55.5 (C5), 52.1 (C36), 48.3 (C17), 47.8 (C9), 46.7 (C19), 46.4 (C31), 43.7 (C18), 42.0 (C14), 39.3 (C38), 39.2 (C8), 38.2 (C1), 37.8 (C4), 37.2 (C10), 34.2 (C21), 33.1 (C29), 32.9 (C7),

TE D

31.7 (C39), 30.5 (C20), 30.5 (C22), 28.2 (C15), 28.2 (C23), 26.0 (C27), 24.1 (C30), 23.7 (C2), 23.5 (C11), 23.1 (C16), 21.4 (C43), 20.2 (C40), 18.4 (C6), 17.1 (C26), 16.8 (C24), 15.5 (C25), 13.9 (C41) ppm; MS (ESI): m/z (%) = 691.3 ([M+H]+, 100), 713.5 ([M+Na]+, 15), 1381.3 ([2M+H]+, 40), 1403.5 ([2M+Na]+, 55); analysis calculated for C43H66N2O5 (690.99):

EP

C 74.74, H 9.63, N 4.05; found: C 74.50, H 9.84, N 3.77.

4.3.16 (3β) 3-Hydroxy-N-(2-butylamino-2-oxoethyl)-N-(furan-2-ylmethyl)-olean-12-en-28-

AC C

carboxamide (3d)

Compound 3d was prepared according to GP C by deacetylation of 3c (70 mg, 0.10 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 7:3) gave 3d (57 mg, 87 %) as a colorless solid; RF = 0.49 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 123–125 °C; [α]D = +5.70° (c = 0.32, CHCl3); IR (KBr): v = 3440s, 2944vs, 2872s, 1666s 1622s, 1542m, 1466m, 1400m, 1386m, 1378m, 1364m, 1282w, 1250m, 1178m, 1146m, 1048m, 1030m, 1012m, 1004m, 734m cm–1; 1H NMR (400 MHz, CDCl3): δ = 7.35 (dd, J = 1.8, 0.7 Hz, 1H, H–33), 6.33 (dd, J = 3.2, 1.9 Hz, 1H, H–34), 6.28 (d, J = 3.2 Hz, 1H, H–35), 6.22 (dd, J = 5.0, 5.0 Hz, 1H, NH), 5.26 (dd, J = 3.1, 3.1 Hz, 1H, H–12), 4.83 (br s, 1H, H– 31a), 4.69 (d, J = 16.0 Hz, 1H, H–31b), 4.15 (d, J = 15.4 Hz, 1H, H–36a), 3.70 (br s, 1H, H– 27

ACCEPTED MANUSCRIPT 36b), 3.25–3.04 (m, 4H, H–3 + H–38a + H–38b + H–18), 2.15 (ddd, J = 14.3, 14.3, 2.9 Hz, 1H, H–16a), 1.97–1.66 (m, 6H, H–11a + H–11b + H–16b + H–22a + H–22b + H–19a), 1.66– 1.57 (m, 4H, H–15a + H–2a + H–2b + H–1a), 1.57–1.49 (m, 2H, H–6a + H–9), 1.46–1.35 (m, 5H, H–7a + H–39a + H–39b + H–21a + H–6b), 1.36–1.26 (m, 2H, H–40a + H–40b), 1.26– 1.15 (m, 3H, H–7b + H–21b + H–19b), 1.13 (s, 3H, H–27), 1.06 (ddd, J = 13.8, 3.0, 3.0 Hz,

RI PT

1H, H–15b), 0.98 (s, 3H, H–23), 0.97–0.94 (m, 1H, H–1b), 0.93 (s, 3H, H–30), 0.90 (s, 3H, H–29), 0.89 (s, 3H, H–25), 0.89 (t, J = 7.4 Hz, 3H, C41), 0.77 (s, 3H, H–24), 0.75–0.71 (m, 1H, H–5), 0.70 (s, 3H, H–26) ppm;

13

C NMR (100 MHz, CDCl3): δ = 177.7 (C28), 169.3

(C37), 150.0 (C32), 144.9 (C13), 142.6 (C33), 121.9 (C12), 110.7 (C34), 109.4 (C35), 79.2

SC

(C3), 55.4 (C5), 52.1 (C36), 48.3 (C17), 47.9 (C9), 46.8 (C19), 46.4 (C31), 43.7 (C18), 42.1 (C14), 39.3 (C8), 39.2 (C38), 38.9 (C4), 38.6 (C1), 37.2 (C10), 34.2 (C21), 33.1 (C29), 33.0 (C7), 31.7 (C39), 30.5 (C22), 30.5 (C20), 28.2 (C23), 28.2 (C15), 27.4 (C2), 26.0 (C27), 24.1

M AN U

(C30), 23.5 (C11), 23.1 (C16), 20.2 (C40), 18.5 (C6), 17.1 (C26), 15.7 (C24), 15.5 (C25), 13.9 (C41) ppm; MS (ESI): m/z (%) = 649.3 ([M+H]+, 40), 671.5 ([M+Na]+, 15), 1297.3 ([2M+H]+, 15), 1319.5 ([2M+Na]+, 100); analysis calculated for C41H64N2O4 (648.96): C 75.88, H 9.94, N 4.32; found: C 75.57, H 10.14, N 4.09.

(3e)

TE D

4.3.17 (3β) 3-Acetoxy-N-(2-butylamino-2-oxoethyl)-N-benzyl-olean-12-en-28-carboxamide

Compound 3e was prepared according to GP A by reaction of OA (300 mg, 0.66 mmol) with benzylamine (86 µL, 0.79 mmol), n–butyl isocyanide (69 µL, 0.66 mmol) and

EP

paraformaldehyde (20 mg, 0.67 mmol), followed by a reaction according to GP B and column chromatography (silica gel, n-hexane/ethyl acetate,8:2) to yield 3e (240 g, 59 %) as a colorless solid; RF = 0.67 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3);

AC C

mp = 115–116 °C; [α]D = +25.27° (c = 0.36, CHCl3); IR (KBr): v = 3342m, 2950vs, 2874s, 1734s, 1684s, 1636s, 1620s, 1540m, 1466s, 1456s, 1390s, 1366s, 1246vs, 1202m, 1180m, 1076w, 1028s, 1004m, 986m, 968m, 730m, 696m cm–1; 1H NMR (500 MHz, CDCl3): δ = 7.36–7.31 (m, 1H, H–34), 7.29–7.25 (m, 1H, H–35), 7.21–7.18 (m, 1H, H–33), 6.22 (br s, 1H, NH), 5.20 (br s, 1H, H–12), 4.95 (d, J = 15.3 Hz, 1H, H–31a), 4.83 (d, J = 15.3 Hz, 1H, H– 31b), 4.52–4.44 (m, 1H, H–3), 4.03 (d, J = 12.7 Hz, 1H, H–36a), 3.70 (br s, 1H, H–36b), 3.21 (dddd, J = 13.3, 6.9, 6.9, 6.9 Hz, 1H, H–38a), 3.11 (dddd, J = 13.3, 6.8, 6.8, 6.8 Hz, 1H, H– 38b), 3.04 (dd, J = 10.4, 1.9 Hz, 1H, H–18), 2.14–2.06 (m, 1H, H–16a), 2.04 (s, 3H, H–43), 1.97–1.81 (m, 2H, H–11a + H–11b), 1.80–1.65 (m, 5H, H–16b + H–15a + H–22a + H–22b +H–19a), 1.65–1.58 (m, 3H, H–2a + H–2b + H–1a), 1.58–1.50 (m, 2H, H–9 + H–6a), 1.47– 28

ACCEPTED MANUSCRIPT 1.36 (m, 2H, H–39a + H–7a), 1.37–1.27 (m, 4H, H–40a + H–40b + H–39b + H–21a), 1.27– 1.14 (m, 3H, H–19b + H–21b + H–7b), 1.11 (s, 3H, H–27), 1.08–0.96 (m, 2H, H–1b + H– 15b), 0.93 (s, 3H, H–25), 0.92 (s, 3H, H–30), 0.89 (t, J = 7.3 Hz, 3H, H–41), 0.89 (s, 3H, H– 29), 0.86 (s, 3H, H–23), 0.86 (s, 3H, H–24), 0.85–0.81 (m, 1H, H–5), 0.79 (s, 3H, H–26) ppm; 13

C NMR (125 MHz, CDCl3): δ = 178.2 (C28), 171.1 (C42), 169.3 (C37), 144.9 (C13), 136.6

RI PT

(C32), 128.8 (34), 127.7 (C35), 127.3 (C33), 121.7 (C12), 81.1 (C3), 55.5 (C5), 53.1 (C31), 52.0 (C36), 48.3 (C17), 47.8 (C9), 46.9 (C19), 43.6 (C18), 42.1 (C14), 39.4 (C38), 39.2 (C8), 38.2 (C4), 37.8 (C1), 37.1 (C10), 34.2 (C21), 33.0 (C29), 32.9 (C7), 31.7 (C39), 30.6 (C20), 30.5 (C22), 28.3 (C15), 28.2 (C23), 25.9 (C27), 24.2 (C30), 23.7 (C2), 23.5 (C11), 23.3 (C16), 21.4 (C43), 20.2 (C40), 18.4 (C6), 17.4 (C26), 16.8 (C24), 15.5 (C25), 13.9 (C41)

SC

ppm; MS (ESI): m/z (%) = 701.3 ([M+H]+, 85), 723.5 ([M+Na]+, 30), 1401.3 ([2M+H]+, 70),

4.00; found: C 76.88, H 10.01, N 3.76.

M AN U

1424.5 ([2M+Na]+, 100); analysis calculated for C45H68N2O4 (701.03): C 77.10, H 9.78, N

4.3.18 (3β) 3-Hydroxy-N-(2-butylamino-2-oxoethyl)-N-benzyl-olean-12-en-28-carboxamide (3f)

Compound 3f was prepared according to GP C by deacetylation of compound 3e (100 mg,

TE D

0.14 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 7:3) yielded 3f (85 mg, 98 %) as a colorless solid; RF = 0.52 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 130–133 °C ; [α]D = +20.82° (c = 0.34, CHCl3); IR (KBr): v = 3442s, 3432s, 3946vs, 2872m, 1666s, 1618s, 1546m, 1496w, 1464m, 1456m, 1386m, 1364m, 1284w,

EP

1226w, 1202m, 1182m, 1048w, 1030w, 1002m, 732m cm–1; 1H NMR (500 MHz, CDCl3): δ = 7.36–7.31 (m, 1H, H–34), 7.29–7.25 (m, 1H, H–35), 7.21–7.18 (m, 1H, H–33), 6.25 (br s, 1H, NH), 5.20 (br s, 1H, H–12), 4.96 (d, J = 15.6 Hz, 1H, H–31a), 4.84 (d, J = 13.9 Hz, 1H, H–

AC C

31b), 4.03 (d, J = 13.4 Hz, 1H, H–36a), 3.71 (d, J = 7.0 Hz, 1H, H–36b), 3.25–3.17 (m, 2H, H–38a + H–3), 3.12 (dddd, J = 13.0, 6.6, 6.6, 6.6 Hz, 1H, H–38b), 3.04 (dd, J = 10.5, 1.9 Hz, 1H, H–18), 2.09 (ddd, J = 15.1, 15.1, 3.8 Hz, 1H, H–16a), 1.97–1.81 (m, 2H, H–11a + H– 11b), 1.79–1.65 (m, 5H, H–22a + H–22b + H–16b + H–19a + H–15a), 1.63–1.56 (m, 3H, H– 2a + H–2b + H–1a), 1.56–1.51 (m, 2H, H–9 + H–6a), 1.46–1.36 (m, 4H, H–6b + H–7a + H– 39a + H–39b), 1.36–1.27 (m, 3H, H–40a + H–40b + H–21a), 1.27–1.13 (m, 3H, H–21b + H– 7b + H–19b), 1.11 (s, 3H, H–27), 1.02–0.98 (m, 1H, H–15b), 0.98 (s, 3H, H–23), 0.98–0.92 (m, 1H, H–1b), 0.91 (s, 3H, H–30), 0.90 (t, J = 7.2 Hz, 3H, H–41), 0.90 (s, 3H, H–25), 0.88 (s, 3H, H–29), 0.79 (s, 3H, H–26), 0.78 (s, 3H, H–24), 0.74–0.70 (m, 1H, H–5) ppm;

13

C

NMR (125 MHz, CDCl3): δ = 178.2 (C28), 169.3 (C37), 144.9 (C13), 136.6 (C32), 128.8 29

ACCEPTED MANUSCRIPT (C34), 127.7 (C35), 127.3 (C33), 121.8 (C12), 79.1 (C3), 55.4 (C5), 53.1 (C31), 51.9 (C36), 48.3 (C17), 47.9 (C9), 47.0 (C19), 43.5 (C18), 42.1 (C14), 39.4 (C38), 39.2 (C8), 38.9 (C4), 38.5 (C1), 37.2 (C10), 34.2 (C21), 33.0 (C29), 33.0 (C7), 31.7 (C39), 30.5 (C20), 30.5 (C22), 28.3 (C15), 28.2 (C23), 27.3 (C2), 26.0 (C27), 24.2 (C30), 23.5 (C11), 23.3 (C16), 20.2 (C40), 18.5 (C6), 17.4 (C26), 15.7 (C24), 15.5 (C25), 13.9 (C41) ppm; MS (ESI): m/z (%) =

RI PT

659.4 ([M+H]+, 60), 681.6 ([M+Na]+, 10), 1317.4 ([2M+H]+, 20), 1339.7 ([2M+Na]+, 100); analysis calculated for C43H66N2O3 (659.00): C 78.37, H 10.09, N 4.25; found: C 78.04, H 10.31, N 4.07.

SC

4.3.19 (3β) 3-Acetoxy-N-(2-benzyl-2-oxoethyl)-N-butanyl-olean-12-en-28-carboxamide (4a) Compound 4a was prepared according to GP A by reaction of OA (250 mg, 0.55 mmol) with butylamine (65 µL, 0.66 mmol), benzyl isocyanide (66 µL, 0.54 mmol) and paraformaldehyde

M AN U

(16 mg, 0.53 mmol). After acetylation according to GP B and column chromatography (silica gel, n-hexane/ethyl acetate, 8:2) 4a (246 mg, 64 %) was obtained as a colorless solid; RF = 0.70 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 108–110 °C; [α]D = –12.43° (c = 0.32, CHCl3); IR (KBr): v = 3448vs, 2948s, 2874m, 1734m, 1684m, 1670s, 1654s, 1636s, 1540m, 1534m, 1466m, 1456m, 1388m, 1370m, 1246s, 1110w, 1028m,

TE D

1006w cm–1; 1H NMR (400 MHz, CDCl3): δ = 7.30–7.25 (m, 1H, H–40), 7.25–7.19 (m, 2H, H–39 + H–41), 7.03 (s, 1H, NH), 5.23 (dd, J = 3.5 Hz, 1H, H–12), 4.55–4.45 (m, 2H, H–3 + H–37a), 4.29–4.16 (m, 2H, H–37b + H–35a), 3.74–3.55 (m, 2H, H–35b + H–31a), 3.35 (ddd, J = 13.9, 13.9, 4.4 Hz, 1H, H–31b), 2.99 (dd, J = 13.6, 2.3 Hz, 1H, H–18), 2.10 (ddd, J =

EP

14.0, 14.0, 2.5 Hz, 1H, H–16a), 2.03 (s, 1H, H–43), 1.94–1.79 (m, 2H, H–11a + H–11b), 1.72–1.59 (m, 7H, H–16b + H–2a + H–2b + H–22a + H–22b + H–1a + H–19a), 1.60–1.46 (m, 5H, H–9 + H–32a + H–32b + H–15a + H–6a), 1.45–1.24 (m, 5H, H–6b + H–7b + H–21b +

AC C

H–33a + H–33b), 1.23–1.13 (m, 3H, H–21b + H–7b + H–19b), 1.11 (s, 3H, H–27), 1.09–0.96 (m, 2H, H–15b + H–1b), 0.93 (t, J = 7.3 Hz, 3H, H–34), 0.89 (s, 3H, H–25), 0.89 (s, 3H, H– 29), 0.87 (s, 3H, H–30), 0.85 (s, 3H, H–23), 0.84 (s, 3H, H–24), 0.83–0.79 (m, 1H, H–5), 0.66 (s, 3H, H–26) ppm; 13C NMR (100 MHz, CDCl3): δ = 177.8 (C28), 171.1 (C42), 170.3 (C36), 144.8 (C13), 138.4 (C38), 128.7 (40), 127.7 (C41), 127.4 (C39), 121.7 (C12), 81.0 (C3), 55.5 (C5), 53.0 (C35), 50.1 (C31), 48.0 (C17), 47.8 (C9), 46.8 (C19), 43.6 (C18), 43.4 (C37), 42.1 (C14), 39.2 (C8), 38.2 (C1), 37.8 (C4), 37.1 (C10), 34.2 (C21), 33.0 (C29), 32.8 (C7), 30.5 (C22), 30.2 (C20), 30.0 (C32), 28.3 (C15), 28.2 (C23), 25.9 (C27), 24.2 (C30), 23.6 (C2), 23.5 (C11), 22.8 (C16), 21.4 (C43), 20.3 (C33), 18.3 (C6), 17.1 (C26), 16.8 (C24), 15.5 (C25), 14.0 (C34) ppm; MS (ESI): m/z (%) = 701.3 ([M+H]+, 100), 723.5 ([M+Na]+, 15), 30

ACCEPTED MANUSCRIPT 1401.3 ([2M+H]+, 15), 1423.5 ([2M+Na]+, 20); analysis calculated for C45H68N2O4 (701.03): C 77.10, H 9.78, N 4.00; found: C 76.94, H 9.96, N 3.74.

4.3.20 (3β) 3-Hydroxy-N-(2-benzyl-2-oxoethyl)-N-butanyl-olean-12-en-28-carboxamide (4b) Compound 4b was prepared according to GP C by deacetylation of 4a (90 mg, 0.13 mmol).

RI PT

Column chromatography (silica gel, n-hexane/ethyl acetate, 6:4) gave 4b (84 mg, 99 %) as a colorless solid; RF = 0.49 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 119–120 °C; [α]D = –10.76° (c = 0.33, CHCl3); IR (KBr): v = 3442vs, 2946s, 2872m, 1664m, 1616m, 1540m, 1498w, 1464m, 1456m, 1402w, 1386m, 1364w, 1280w, 1248w,

SC

1204w, 1182m, 1048m, 1030m, 1000m cm–1; 1H NMR (400 MHz, CDCl3): δ = 7.30–7.26 (m, 1H, H–40), 7.24–7.20 (m, 2H, H–39 + H–41), 7.06 (br s, 1H, NH), 5.23 (dd, J = 3.4, 3.4 Hz, 1H, H–12), 4.50 (dd, J = 14.8, 6.7 Hz, 1H, H–37a), 4.29–4.16 (m, 2H, H–37b + H–35a),

M AN U

3.70–3.55 (m, 2H, H–35b + H–31a), 3.40–3.30 (m, 1H, H–31b), 3.19 (dd, J = 11.2, 4.5 Hz, 1H, H–3), 2.99 (dd, J = 13.9, 1.6 Hz, 1H, H–18), 2.10 (ddd, J = 14.6, 14.6, 3.0 Hz, 1H, H– 16a), 1.93–1.79 (m, 2H, H–11a + H–11b), 1.71–1.62 (m, 4H, H–16b + H–19a + H–22a + H– 22b), 1.62–1.48 (m, 8H, H–32a + H–32b + H–15a + H–2a + H+2b + H–1a + H–6a + H–9), 1.43–1.23 (m, 5H, H–6b + H–33a + H–33b + H–7a + H–21a), 1.23–1.12 (m, 3H, H–19b + H–

TE D

21b + H–7b), 1.12 (s, 3H, H–27), 1.08–1.02 (m, 1H, H–15b), 0.97 (s, 3H, H–23), 0.96–0.90 (m, 1H, H–1b), 0.93 (t, J = 7.3 Hz, 3H, H–34), 0.88 (s, 3H, H–29), 0.87 (s, 6H, H–30 + H– 25), 0.77 (s, 3H, H–24), 0.73–0.68 (m, 1H, H–5), 0.66 (s, 3H, H–26) ppm;

13

C NMR (100

MHz, CDCl3): δ = 177.8 (C28), 170.3 (C36), 144.8 (C13), 138.4 (C38), 128.7 (C40), 127.7

EP

(C41), 127.4 (C39), 121.8 (C12), 79.1 (C3), 55.4 (C5), 53.0 (C35), 50.1 (C31), 48.0 (C17), 47.8 (C9), 46.8 (C19), 43.6 (C18), 43.4 (C37), 42.1 (C14), 39.2 (C8), 38.9 (C4), 38.5 (C1), 37.2 (C10), 34.2 (C21), 33.0 (C29), 32.9 (C7), 30.5 (C22), 30.2 (C20), 30.0 (C32), 28.3

AC C

(C15), 28.2 (C23), 27.3 (C2), 26.0 (C27), 24.3 (C30), 23.5 (C11), 22.8 (C16), 20.3 (C33), 18.4 (C6), 17.1 (C26), 15.7 (C24), 15.5 (C25), 14.0 (C34) ppm; MS (ESI): m/z (%) = 659.3 ([M+H]+, 60), 681.5 ([M+Na]+, 20), 713.4 ([M+Na+MeOH]+, 15), 1317.5 ([2M+H]+, 15), 1339.5 ([2M+Na]+, 100); analysis calculated for C43H66N2O3 (659.00): C 78.37, H 10.09, N 4.25; found: C 78.05, H 10.24, N 4.03.

4.3.21

(3β)

3-Acetoxy-N-(2-benzylamino-2-oxoethyl)-N-(1-methylethyl)-olean-12-en-28-

carboxamide (4c) Compound 4c was prepared according to GP A by reaction of OA (200 mg, 0.44 mmol) with isopropylamine (45 µL, 0.53 mmol), benzyl isocyanide (52 µL, 0.44 mmol) and 31

ACCEPTED MANUSCRIPT paraformaldehyde (14 mg, 47 mmol). Acetylation and column chromatography (silica gel, nhexane/ethyl acetate, 7:3) gave 4c (268 mg, 89 %) as a colorless solid; RF = 0.67 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 128–130 °C; [α]D = –38.41° (c = 0.32, CHCl3); IR (KBr): v = 3446vs, 2948m, 2878m, 1734m, 1676m, 1654m, 1628m, 1534w, 1466w, 1456w, 1394w, 1370w, 1246m, 1174w, 1158w, 1028w, 1006w cm–1; 1H NMR

RI PT

(400 MHz, CDCl3): δ = 7.31–7.24 (m, 2H, H–39), 7.24–7.18 (m, 3H, H–38 + H–40), 5.23 (dd, J = 3.4, 3.4 Hz, 1H, H–12), 4.60–4.51 (m, 2H, H–31 + H–36a), 4.47 (dd, J = 9.4, 6.5 Hz, 1H, H–3), 4.16 (m, 2H, H–34a + H–36b), 3.58 (d, J = 14.2 Hz, 1H, H–34b), 3.00 (dd, J = 13.9, 2.5 Hz, 1H, H–18), 2.13 (ddd, J = 14.4, 14.4, 2.4 Hz, 1H, H–16a), 2.03 (s, 3H, H–42), 1.96–1.79

SC

(m, 2H, H–11a + H–11b), 1.74–1.64 (m, 3H, H–16b + H–22a + H–19a), 1.63–1.44 (m, 7H, H–2a + H–2b + H–22b + H–15a + H–1a + H–9 + H–6a), 1.43–1.25 (m, 3H, H–6b + H–21a + H–7a), 1.23–1.14 (m, 3H, H–21b + H–7b + H–19b),1.22 (d, J = 6.4 Hz, 3H, H–33), 1.19 (d, J

M AN U

= 6.5 Hz, 3H, H–32), 1.12 (s, 3H, H–27), 1.10–1.05 (m, 1H, H–15b), 1.05–0.97 (m, 1H, H– 1b), 0.90 (s, 3H, H–25), 0.89 (s, 3H, H–29), 0.85 (s, 3H, H–30), 0.85 (s, 3H, H–23), 0.84 (s, 3H, H–24), 0.83–0.79 (m, 1H, H–5), 0.68 (s, 3H, H–26) ppm; 13C NMR (100 MHz, CDCl3): δ = 177.7 (C28), 171.6 (C35), 171.1 (C41), 145.0 (C13), 138.6 (C37), 128.6 (C39), 127.7 (C40), 127.3 (C38), 121.5 (C12), 81.1 (C3), 55.5 (C5), 49.2 (C31), 48.3 (C17), 47.8 (C34),

TE D

47.6 (C9), 46.9 (C19), 43.9 (C18), 43.4 (C36), 42.1 (C14), 39.2 (C8), 38.2 (C1), 37.8 (C4), 37.1 (C10), 34.3 (C21), 33.0 (C29), 32.8 (C7), 30.4 (C20), 29.9 (C22), 28.4 (C15), 28.2 (C23), 25.9 (C27), 24.1 (C30), 23.6 (C2), 23.5 (C11), 22.9 (C16), 21.4 (C42), 21.3 (C32), 20.3 (C33), 18.3 (C6), 17.1 (C26), 16.8 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 687.3

EP

([M+H]+, 100), 709.5 ([M+Na]+, 35), 1374.3 ([2M+H]+, 20), 1395.4 ([2M+Na]+, 45); analysis calculated for C44H66N2O4 (687.01): C 76.92, H 9.68, N 4.08; found: C 76.71, H 9.85, N 3.82. (3β)

3-Hydroxy-N-(2-benzylamino-2-oxoethyl)-N-(1-methylethyl)-olean-12-en-28-

AC C

4.3.22

carboxamide (4d)

Compound 4d was prepared according to GP C by deacetylation of 4c (100 mg, 0.16 mmol). Column chromatography (silica gel, ethyl acetate/chloroform, 2:8) gave 4d (85 mg, 80 %) as a colorless solid; mp = 129–130 °C; RF = 0.48 (silica gel, toluene/ethyl acetate/n– heptane/formic acid, 80:30:10:3); [α]D = –37.47° (c = 0.60, CHCl3); IR (KBr): v = 3440s, 2944vs, 2874s, 1668s, 1608s, 1534s, 1456s, 1388s, 1328m, 1280m, 1242m, 1176m, 1074m, 1030s, 1000m cm–1; 1H NMR (500 MHz, CDCl3): δ = 7.30–7.19 (m, 5H, H–38 + H–39 + H– 40), 5.24 (dd, J = 3.5, 3.5 Hz, 1H, H–12), 4.61–4.50 (m, 2H, H–31 + H–36a), 4.16 (m, 2H, H– 34a + H–36b), 3.60 (br s, 1H, H–34b), 3.19 (dd, J = 11.3, 4.4 Hz, 1H, H–3), 3.01 (dd, J = 32

ACCEPTED MANUSCRIPT 12.9, 1.2 Hz, 1H, H–18), 2.13 (ddd, J = 14.2, 14.2, 2.2 Hz, 1H, H–16a), 1.96–1.80 (m, 2H, H– 11a + H–11b), 1.74–1.63 (m, 3H, H–16b + H–19a + H–22a), 1.62–1.46 (m, 7H, H–1a + H–2a + H–2b + H–15a + H–22b + H–6a + H–9), 1.44–1.28 (m, 3H, H–6b + H–7a + H–21a), 1.23 (d, J = 6.5 Hz, 3H, H–32), 1.27–1.14 (m, 3H, H–7b + H–21b + H–19b), 1.19 (d, J = 6.6 Hz, 3H, H–33), 1.13 (s, 3H, H–27), 1.12–1.06 (m, 1H, H–15b), 0.97 (s, 3H, H–23), 0.92 (m, 1H,

RI PT

H–1b), 0.89 (s, 3H, H–29), 0.88 (s, 3H, H–25), 0.86 (s, 3H, H–30), 0.77 (s, 3H, H–24), 0.74– 0.69 (m, 1H, H–5), 0.68 (s, 3H, H–26) ppm; 13C NMR (125 MHz, CDCl3): δ = 177.7 (C28), 171.7 (C35), 145.0 (C13), 138.6 (C37), 128.6 (C39), 127.7 (C40), 127.3 (C38), 121.6 (C12), 79.1 (C3), 55.5 (C5), 49.2 (C31), 48.3 (C17), 47.9 (C9), 47.6 (C34), 46.9 (C19), 43.9 (C18),

SC

43.4 (C36), 42.2 (C14), 39.2 (C8), 38.9 (C4), 38.5 (C1), 37.2 (C10), 34.3 (C21), 33.0 (C29), 32.9 (C7), 30.4 (C22), 30.0 (C20), 28.4 (C15), 28.2 (C23), 27.3 (C2), 25.9 (C27), 24.1 (C30), 23.5 (C11), 22.9 (C16), 21.3 (C32), 20.3 (C33), 18.4 (C6), 17.1 (C26), 15.7 (C24), 15.5 (C25)

M AN U

ppm; MS (ESI): m/z (%) = 645.3 ([M+H]+, 100), 667.5 ([M+Na]+, 10), 987.7 ([3M+K+H]2+, 10), 1290.4 ([2M+H]+, 62), 1311,5 ([2M+Na]+, 90); analysis calculated for C42H64N2O3 (644.97): C 78.21, H 10.00, N 4.34; found: C 77.98, H 10.29, N 4.17.

4.3.23

(3β)

3-Acetoxy-N-(2-benzylamino-2-oxoethyl)-N-(prop-2-enyl)-olean-12-en-28-

TE D

carboxamide (4e)

Compound 4e was prepared according to GP A by reaction of OA (200 mg, 0.44 mmol) with allylamine (42 µL, 0.56 mmol), benzyl isocyanide (52 µL, 0.44 mmol) and paraformaldehyde (14 mg, 0.47 mmol). Acetylation and column chromatography (silica gel, n-hexane/ethyl

EP

acetate, 7:3) gave 4e (240 mg, 80 %) as a colorless solid; RF = 0.63 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 122–124 °C; [α]D = –12.73° (c = 0.31, CHCl3); IR (KBr): v = 3442vs, 2946s, 2876m, 1734s, 1684m, 1670m, 1654m, 1636s, 1624s,

AC C

1540m, 1534m, 1498w, 1464m, 1456m, 1436w, 1390m, 1370m, 1246s, 1204m, 1182m, 1164w, 1028m, 1004m, 988m cm–1; 1H NMR (400 MHz, CDCl3): δ = 7.31–7.20 (m, 5H, H– 38 + H–39 + H–40), 6.82 (br s, 1H, NH), 5.74 (ddd, J = 17.1, 9.8, 5.6 Hz, 1H, H–32), 5.31– 5.25 (m, 2H, H–33a + H–33b), 5.24 (dd, J = 3.5, 3.5 Hz, 1H, H–12), 4.52–4.45 (m, 2H, H–3 + H–36a), 4.38 (d, J = 14.5 Hz, 1H, H–31a), 4.28 (dd, J = 14.8, 5.0 Hz, 1H, H–36b), 4.19 (d, J = 14.7 Hz, 1H, H–34a), 4.03 (dd, J = 16.2, 5.8 Hz, 1H, H–31b), 3.63 (d, J = 12.7 Hz, 1H, H– 34b), 3.00 (dd, J = 14.1, 3.0 Hz, 1H, H–18), 2.10 (ddd, J = 15.3, 14.9, 3.4 Hz, 1H, H–16), 2.03 (s, 3H, H–42), 1.95–1.78 (m, 2H, H–11a + H–11b), 1.75–1.65 (m, 2H, H–19a + H–16b), 1.66–1.47 (m, 9H, H–22a + H–2a + H–2b + H–19a + H–1a + H–9 + H–22b + H–15a + H– 6a), 1.46–1.26 (m, 3H, H–7a + H–6b + H–21a), 1.24–1.12 (m, 3H, H–7b + H–21b + H–19b), 33

ACCEPTED MANUSCRIPT 1.11 (s, 3H, H–27), 1.07–0.98 (m, 2H, H–1b + H–15b), 0.90 (s, 3H, H–25), 0.89 (s, 3H, H– 29), 0.87 (s, 3H, H–30), 0.85 (s, 3H, H–23), 0.84 (s, 3H, H–24), 0.84–0.80 (m, 1H, H–5), 0.68 (s, 3H, H–26) ppm; 13C NMR (100 MHz, CDCl3): δ =177.8 (C28), 171.1 (C41), 169.7 (C35), 144.8 (C13), 138.4 (C37), 132.4 (C32), 128.7 (C39), 127.8 (C40), 127.5 (C38), 121.8 (C12), 119.4 (C33), 81.1 (C3), 55.5 (C5), 52.2 (C31), 51.6 (C34), 48.0 (C17), 47.8 (C9), 46.7 (C19),

RI PT

43.6 (C18), 43.4 (C36), 42.0 (C14), 39.3 (C8), 38.2 (C1), 37.8 (C4), 37.1 (C10), 34.1 (C21), 33.0 (C29), 32.8 (C7), 30.5 (C22), 30.3 (C20), 28.3 (C15), 28.2 (C23), 25.9 (C27), 24.2 (C30), 23.6 (C2), 23.5 (C11), 22.7 (C16), 21.4 (C42), 18.3 (C6), 17.1 (C26), 16.8 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 685.3 ([M+H]+, 95), 707.5 ([M+Na]+, 35), 1370.4

77.15, H 9.42, N 4.09; found: C 76.94, H 9.61, N 3.92.

(3β)

3-Hydroxy-N-(2-benzylamino-2-oxoethyl)-N-(prop-2-enyl)-olean-12-en-28-

M AN U

4.3.24

SC

([2M+H]+, 15), 1391.5 ([2M+Na]+, 50); analysis calculated for C44H64N2O4 (684.99): C

carboxamide (4f)

Compound 4f was prepared according to GP C by deacetylation of 4e (110 mg, 0.16 mmol). Column chromatography (silica gel, ethyl acetate/chloroform, 2:8) gave 4f (85 mg, 82 %) as a colorless solid; RF = 0.51 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 114–115 °C; [α]D = –12.19° (c = 0.69, CHCl3); IR (KBr): v = 3440s, 3326s, 2944vs,

TE D

2872s, 1670s, 1616s, 1540m, 1454vs, 1388s, 1248m, 1181m, 1030s, 1002m, 920m cm–1; 1H NMR (400 MHz, CDCl3): δ = 7.32–7.20 (m, 5H, H–38 + H–39 + H–40), 6.82 (s, 1H, NH), 5.74 (ddd, J = 17.1, 9.8, 5.6 Hz, 1H, H–32), 5.32–5.21 (m, 3H, H–12 + H–33a + H–33b), 4.49

EP

(dd, J = 14.8, 6.5 Hz, 1H, H–36a), 4.38 (br s, 1H, H–31a), 4.28 (dd, J = 14.7, 5.0 Hz, 1H, H– 36b), 4.19 (d, J = 15.0 Hz, 1H, H–34a), 4.03 (dd, J = 16.3, 5.9 Hz, 1H, H–31b), 3.64 (d, J = 14.8 Hz, 1H, H–34b), 3.19 (dd, J = 10.9, 4.7 Hz, 1H, H–3), 3.00 (dd, J = 14.5, 3.3 Hz, 1H, H–

AC C

18), 2.10 (ddd, J = 14.3, 14.3, 2.9 Hz, 1H, H–16a), 1.96–1.79 (m, 2H, H–11a + H–11b), 1.73– 1.47 (m, 10H, H–19a + H–16b + H–1a + H–20a + H–20b + H–15a + H–2a + H–2b + H–9 + H–6a), 1.45–1.24 (m, 3H, H–6b + H–7a + H–21a), 1.24–1.13 (m, 3H, H–21b + H–7b + H– 19b), 1.12 (s, 3H, H–27), 1.08–0.99 (m, 1H, H–15a), 0.98 (s, 3H, H–23), 0.96–0.89 (m, 1H, H–1b), 0.89 (s, 3H, H–29), 0.87 (s, 6H, H–25 + H–30), 0.77 (s, 3H, H–24), 0.74–0.69 (m, 1H, H–5), 0.68 (s, 3H, H–26) ppm; 13C NMR (100 MHz, CDCl3): δ = 177.8 (C28), 169.7 (C35), 144.8 (C13), 138.4 (C37), 132.4 (C32), 128.7 (C39), 127.8 (C40), 127.5 (C38), 121.9 (C12), 119.4 (C33), 79.1 (C3), 55.4 (C5), 52.2 (C31), 51.6 (C34), 48.0 (C17), 47.8 (C9), 46.7 (C19), 43.6 (C18), 43.4 (C36), 42.0 (C14), 39.3 (C8), 38.9 (C4), 38.5 (C1), 37.2 (C10), 34.2 (C21), 33.0 (C29), 32.9 (C7), 30.5 (C22), 30.3 (C20), 28.3 (C15), 28.2 (C23), 27.3 (C2), 26.0 (C27), 34

ACCEPTED MANUSCRIPT 24.2 (C30), 23.5 (C11), 22.7 (C16), 18.5 (C6), 17.1 (C26), 15.7 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 643.3 ([M+H]+, 25), 665.6 ([M+Na]+, 12), 1308.5 ([2M+Na]+, 100); analysis calculated for C42H62N2O3 (642.95): C 78.46, H 9.72, N 4.36; found: C 78.39, H 9.97, N 4.18.

RI PT

4.3.25 (3β) 3–Acetoxy–N–(2–benzylamino–2–oxoethyl)–N–(furan–2–ylmethyl)–olean–12–en– 28–carboxamide (4g)

Compound 4g was prepared according to GP A by reaction of OA (200 mg, 0.44 mmol) with furfurylamine (47 µL, 0.53 mmol), benzyl isocyanide (53 µL, 0.44 mmol) and

SC

paraformaldehyde (14 mg, 0.47 mmol), followed by acetylation. Column chromatography (silica gel, n-hexane/ethyl actetate, 8:2) gave 4g (235 mg, 74 %) as a colorless solid; RF = 0.68 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 114–116 °C;

M AN U

[α]D = +4.58° (c = 0.31, CHCl3); IR (KBr): v = 3442vs, 2948s, 2876m, 1733m, 1670m, 1640m, 1454s, 1247s, 1028m cm–1; 1H NMR (400 MHz, CDCl3) δ = 7.32–7.19 (m, 6H, H–33 + H–40 + H–41 + H–42), 6.62 (br s, 1H, NH), 6.31 (dd, J = 3.1, 1.9 Hz, 1H, H–34), 6.28– 6.25 (m, 1H, H–35), 5.23 (dd, J = 3.2, 3.2 Hz, 1H, H–12), 4.84 (d, J = 16.0 Hz,1H, H–31a), 4.70 (d, J = 16.0 Hz, 1H, H–31b), 4.51–4.45 (m, 1H, H–3), 4.41 (dd, J = 14.8, 6.2 Hz, 1H, H–

TE D

38a), 4.28 (dd, J = 14.8, 5.2 Hz, 1H, H–38b), 4.20 (d, J = 15.3 Hz, 1H, H–36a), 3.80–3.71 (m, 1H, H–36b), 3.03 (dd, J = 13.9, 2.7 Hz, 1H, H–18), 2.14 (ddd, J = 14.3, 14.3, 2.6 Hz, 1H, H– 16a), 2.03 (s, 3H, H–44), 1.92–1.74 (m, 3H, H–11a + H–11b + H–16b), 1.74–1.62 (m, 2H, H– 22a + H–19a), 1.61–1.47 (m, 7H, H–22b + H–1a + H–15a + H–2a + H–2b + H–9 + H–6a),

EP

1.45–1.28 (m, 3H, H–6b + H–21a + H–7a), 1.24–1.13 (m, 3H, H–19b + H–21b + H–7b), 1.11 (s, 3H, H–27), 1.09–0.95 (m, 2H, H–1b + H–15b), 0.89 (s, 9H, H–29 + H–30 + H–28), 0.85 (s, 3H, H–23), 0.84 (s, 3H, H–24), 0.86–0.78 (m, 1H, H–5), 0.65 (s, 3H, H–26) ppm;

13

C

AC C

NMR (100 MHz, CDCl3) δ = 177.8 (C28), 171.1 (C43), 169.4 (C37), 149.8 (C32), 144.8 (C13), 142.7 (C33), 138.2 (C39), 128.7 (C41), 127.9 (C42), 127.5 (C40), 121.7 (C12), 110.6 (C34), 109.4 (C35), 81.1 (C3), 55.5 (C5), 52.0 (C36), 48.3 (C17), 47.8 (C9), 46.7(C19), 46.4 (C31), 43.7 (C18), 43.5 (C38), 42.0 (C14), 39.2 (C8), 38.2 (C1), 37.8 (C4), 37.1 (C10), 34.2 (C21), 33.0 (C29), 32.8 (C7), 30.5 (C22), 28.2 (C15), 28.2 (C23), 25.9 (C27), 24.2 (C30), 23.6 (C2), 23.5 (C11), 23.1 (C16), 21.4 (C44), 18.4 (C6), 17.1 (C26), 16.8 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 725.4 ([M+H]+, 40), 747.5 ([M+Na]+, 45), 1472.4 ([2M+Na]+, 100); analysis calculated for C46H64N2O5 (725.01): C 76.20, H 8.90, N 3.86; found: C 75.96, H 9.13, N 3.57.

35

ACCEPTED MANUSCRIPT 4.3.26 (3β) 3-Hydroxy-N-(2-benzylamino-2-oxoethyl)-N-(furan-2-ylmethyl)-olean-12-en-28carboxamide (4h) Compound 4h was prepared according to GP C by deacetylation of 4g (147 mg, 0.20 mmol). Column chromatography (silica gel, ethyl acetate/chloroform, 2:8) provided 4h (134 mg, 97 %) as a colorless solid; RF = 0.52 (silica gel, toluene/ethyl acetate/n–heptane/formic acid,

RI PT

80:30:10:3); mp = 121–123 °C; [α]D = +4.92° (c = 0.74, CHCl3); IR (KBr): v = 3432s, 3326s, 2944s, 2874s, 1670s, 1620s, 1540s, 1454s, 1386s, 1248s, 1178s, 1076m, 1030s, 1012s, 950m, 734s, 698s, 598m cm–1; 1H NMR (400 MHz, CDCl3) δ = 7.32–7.19 (m, 6H, H–33 + H–40 + H–41 + H–42), 6.64 (dd, J = 4.3, 4.3 Hz, 1H, NH), 6.31 (dd, J = 3.0, 1.9 Hz, 1H, H–34), 6.27

SC

(d, J = 3.1 Hz, 1H, H–35), 5.23 (dd, J = 3.1, 3.1 Hz, 1H, H–12), 4.84 (d, J = 15.6 Hz, 1H, H– 31a), 4.69 (d, J = 16.0 Hz, 1H, H–31b), 4.41 (dd, J = 14.8, 6.1 Hz, 1H, H–38a), 4.28 (dd, J = 14.7, 5.3 Hz, 1H, H–38b), 4.19 (d, J = 15.4 Hz, 1H, H–36a), 3.75 (d, J = 13.4 Hz, 1H, H–

M AN U

36b), 3.19 (dd, J = 10.8, 4.6 Hz, 1H, H–3), 3.03 (dd, J = 13.4, 1.8 Hz, 1H, H–18), 2.13 (ddd, J = 14.4, 14.4, 2.7 Hz, 1H, H–16a), 1.95–1.80 (m, 2H, H–11a + H–11b), 1.80–1.64 (m, 1H, H– 16b + H–22a + H–19a), 1.65–1.54 (m, 4H, H–15a + H–2a + H–2b + H–1a), 1.55–1.47 (m, 2H, H–9 + H–6a), 1.46–1.25 (m, 3H, H–6b + H–7a + H–21a), 1.21–1.13 (m, 3H, H–7b + H– 21b + H–19b), 1.11 (s, 3H, H–27), 1.05–0.99 (m, 1H, H–15b), 0.97 (s, 3H, H–23), 0.96–0.90 (m, 1H, H–1b), 0.89 (s, 6H, H–29 + H–30), 0.86 (s, 3H, H–25), 0.77 (s, 3H, H–24), 0.74–0.67

TE D

(m, 1H, H–5), 0.65 (s, 3H, H–26) ppm; 13C NMR (100 MHz, CDCl3) δ = 177.7 (C28), 169.3 (C37), 149.8 (C32), 144.8 (C13), 142.6 (C33), 138.2 (C39), 128.7 (C41), 127.9 (C42), 127.5 (C40), 121.8 (C12), 110.6 (C34), 109.4 (C35), 79.1 (C3), 55.4 (C5), 51.9 (C36), 48.3 (C9),

EP

47.8 (C17), 46.7 (C19), 46.4 (C31), 43.7 (C18), 43.5 (C38), 42.0 (C14), 39.2 (C8), 38.9 (C4), 38.5 (C1), 37.2 (C10), 34.2 (C21), 33.0 (C29), 32.9 (C7), 30.4 (C22), 30.4 (C20), 28.2 (C23), 28.2 (C15), 27.3 (C2), 26.0 (C27), 24.2 (C30), 23.5 (C11), 23.1 (C16), 18.5 (C6), 17.1 (C26),

AC C

15.7 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 683.2 ([M+H]+, 50), 689.7 ([M+Li]+, 8), 705.5 ([M+Na]+, 10), 1387.4 ([2M+Na]+, 100); analysis calculated for C44H62N2O4 (682.97): C 77.38, H 9.15, N 4.10; found: C 77.01, H 9.32, N 3.96.

4.3.27 (3β) 3-Acetoxy-N-(2-benzylamino-2-oxoethyl)-N-benzyl-olean-12-en-28-carboxamide (4i) Compound 4i was prepared according to GP A by reaction of OA (200 mg, 0.44 mmol) with benzylamine (58 µL, 0.53 mmol), benzyl isocyanide (52 µL, 0.43 mmol) and paraformaldehyde (14 mg, 0.44 mmol). Acetylation according to GP B and column chromatography (silica gel, n-hexane/ethyl acetate, 7:3) gave 4i (215 mg, 67 %) as a colorless 36

ACCEPTED MANUSCRIPT solid; RF = 0.72 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 131–133 °C; [α]D = +18.56° (c = 0.38, CHCl3); IR (KBr): v = 3432s, 2946vs, 2876m, 1734s, 1684s, 1636s, 1534m; 1496m, 1454s, 1366s, 1246vs, 1202m, 1180m, 1028m, 1004m, 988m, 698m cm–1; 1H NMR (400 MHz, CDCl3): δ = 7.36–7.17 (m, 10H, H–33 + H–34 + H–35 + H– 40 + H–41 + H–42), 6.55 (br s, 1H, NH), 5.24–5.11 (br s, 1H, H–12), 4.96 (d, J = 16.5 Hz,

RI PT

1H, H–31a), 4.85 (d, J = 16.7 Hz, 1H, H–31b), 4.54–4.39 (m, 2H, H–3 + H–38a), 4.28 (dd, J = 14.7, 5.2 Hz, 1H, H–38b), 4.08 (dd, J = 14.9 Hz, 1H, H–36a), 3.76 (d, J = 15.0 Hz, 1H, H– 36b), 3.07–2.94 (m, 1H, H–18), 2.15–2.04 (m, 1H, H–16a), 2.04 (s, 3H, H–44), 1.97–1.78 (m, 2H, H–11a + H–11b), 1.75–1.47 (m, 10H, H–16b + H–22a + H–22b + H–15a + H–19a + H–

SC

1a + H–2a + H–2b + H–6a + H–9), 1.45–1.25 (m, 3H, H–21a + H–7a + H–6b), 1.23–1.12 (m, 3H, H–21b + H–7b + H–19b), 1.10 (s, 3H, H–27), 1.08–0.93 (m, 2H, H–1b + H–15b), 0.92 (s, 3H, H–25), 0.88 (s, 6H, H–29 + H–30), 0.86 (s, 6H, H–23 + H–24), 0.85–0.78 (m, 1H, H–5),

M AN U

0.75 (s, 3H, H–26) ppm; 13C NMR (125 MHz, CDCl3): δ = 178.3 (C28), 171.1 (C43), 169.3 (C37), 144.9 (C13), 138.3 (C39), 136.4 (C32), 128.9 (C41), 128.8 (C34), 127.9 (C42), 127.7 (C35), 127.5 (C40), 127.3 (C33), 121.7 (C12), 81.1 (C3), 55.5 (C5), 53.1 (C31), 51.8 (C36), 48.3 (C17), 47.8 (C9), 46.9 (C19), 43.7 (C18), 43.5 (C38), 42.1 (C14), 39.4 (C8), 38.2 (C1), 37.8 (C4), 37.1 (C10), 34.2 (C21), 33.0 (C29), 32.9 (C7), 30.5 (C20), 30.5 (C22), 28.3 (C15), 28.2 (C23), 25.9 (C27), 24.2 (C30), 23.7 (C2), 23.5 (C11), 23.2 (C16) 21.4 (C44), 18.4 (C6),

TE D

17.3 (C26), 16.8 (C24), 15.6 (C25) ppm; MS (ESI): m/z (%) = 735.3 ([M+H]+, 100), 757.5 ([M+Na]+, 10), 1470.3 ([2M+H]+, 25), 1491.5 ([2M+Na]+, 35); analysis calculated for

EP

C48H66N2O4 (735.05): C 78.43, H 9.05, N 3.81; found: C 78.29, H 9.18, N 3.67. 4.3.28 (3β) 3-Hydroxy-N-(2-benzylamino-2-oxoethyl)-N-benzyl-olean-12-en-28-carboxamide (4j)

AC C

Compound 4j was prepared according to GP C by deacetylation of 4i (110 mg, 0.15 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 7:3) provided 4j (90 mg, 87 %) as a colorless solid; RF = 0.57 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 135–138 °C; [α]D = +23.38° (c = 0.34, CHCl3); IR (KBr): v = 3440s, 1944vs, 2845m, 1671s, 1618s, 1534m, 1496m, 1454s, 1387m, 1331m, 1248m, 1180m, 1029m, 1002m, 730m, 698s cm–1; 1H NMR (500 MHz, CDCl3): δ = 7.36–7.17 (m, 10H, H–33 + H–34 + H–35 + H–40 + H–41 + H–42), 6.61 (br s, 1H, NH), 5.17 (br s, 1H, H–12), 4.97 (d, J = 14.8 Hz, 1H, H–31a), 4.85 (d, J = 15.4 Hz, 1H, H–31b) 4.43 (dd, J = 14.7, 6.2 Hz, 1H, H– 38a), 4.29 (dd, J = 14.7, 5.0 Hz, 1H, H–38b), 4.08 (d, J = 13.9 Hz, 1H, H–36a), 3.76 (br s, 1H, H–36b), 3.20 (dd, J = 11.2, 4.6 Hz, 1H, H–3), 3.06–2.96 (m, 1H, H–18), 2.09 (ddd, J = 37

ACCEPTED MANUSCRIPT 14.2, 14.2, 2.5 Hz, 1H, H–16a), 1.95–1.79 (m, 2H, H–11a + H–11b), 1.75–1.64 (m, 5H, H– 16b + H–22a + H–22b + H–15a + H–19a), 1.63–1.55 (m, 3H, H–2a + H–2b + H–1a), 1.55– 1.49 (m, 2H, H–6a + H–9), 1.43–1.36 (m, 2H, H–6b + H–7a), 1.36–1.25 (m, 1H, H–21a), 1.24–1.11 (m, 3H, H–7b + H–21b + H–19b), 1.10 (s, 3H, H–27), 0.98 (s, 3H, H–23), 0.98– 0.90 (m, 2H, H–1b + H–15b), 0.88 (s, 3H, H–25), 0.88 (s, 6H, H–29 + H–30), 0.78 (s, 3H, H– 13

C NMR (125 MHz, CDCl3): δ

RI PT

24), 0.75 (s, 3H, H–26), 0.74–0.68 (m, 1H, H–5) ppm;

=178.3 (C28), 169.3 (C37), 144.9 (C13), 138.3 (C39), 136.4 (C32), 128.9 (C41), 128.7 (C34), 127.9 (C42), 127.7 (C35), 127.5 (C40), 127.3 (C33), 121.77 (C12), 79.1 (C3), 55.4 (C5), 53.1 (C31), 51.8 (C36), 48.3 (C17), 47.8 (C9), 47.0 (C19), 43.6 (C18), 43.5 (C38), 42.1 (C14),

SC

39.3 (C8), 38.9 (C4), 38.5 (C1), 37.2 (C10), 34.2 (C21), 33.0 (C29), 33.0 (C7), 30.5 (C20), 30.5 (C22), 28.3 (C15), 28.2 (C23), 27.3 (C2), 25.9 (C27), 24.2 (C30), 23.5 (C11), 23.2 (C16), 18.5 (C6), 17.4 (C26), 15.7 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 693.3

M AN U

([M+H]+, 100), 715.5 ([M+Na]+, 10), 1386.3 ([2M+H]+, 20), 1408.4 ([2M+Na]+, 60); analysis calculated for C46H64N2O3 (693.01): C 79.72, H 9.31, N 4.04; found: C 79.56, H 9.50, N 3.87. 4.3.29 (3β) 3-Acetoxy-methyl-2-(2-(N-(prop-2-enyl)-olean-12-en-28-carboxamido)acetamido) acetate (5a)

TE D

Compound 5a was prepared according to GP A by reaction of OA (200 mg, 0.44 mmol) with allylamine (48 µL, 0.64 mmol), methyl isocyanoacetate (39 µL, 0.44 mmol) and paraformaldehyde (14 mg, 0.44 mmol). Acetylation according to GP B and column chromatography (silica gel, n-hexane/ethyl acetate, 8:2) gave 5a (150 mg, 51 %) as a

EP

colorless solid; RF = 0.33 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 102–103 °C; [α]D = –12.36° (c = 0.38, CHCl3); IR (KBr): v = 3384m, 2948s, 2876m, 1735s, 1690s, 1638m, 1619m, 1529m, 1458m, 1438m, 1370s, 1247vs, 1205s, 1181s, 1028m,

AC C

1005m, 989m cm–1; 1H NMR (500 MHz, CDCl3): δ = 6.85 (dd, J = 4.6 Hz, 1H, NH), 5.76 (ddd, J = 16.5, 10.9, 5.8 Hz, 1H, H–32), 5.28–5.22 (m, 3H, H–12 + H–33), 4.49–4.45 (m, 1H, H–3), 4.37 (d, J = 13.5 Hz, 1H, H–31a), 4.14 (d, J = 14.4 Hz, 1H, H–34a), 4.04 (m, 2H, H– 31b + H–36a), 3.90 (dd, J = 18.1, 4.8 Hz, 1H, H–36b), 3.71 (s, 3H, H–38), 3.66 (d, J = 13.2 Hz, 1H, H–34b), 3.03 (dd, J = 12.1, 2.9 Hz, 1H, H–18), 2.12 (ddd, J = 14.5, 14.5, 2.3 Hz, 1H, H–16a), 2.02 (s, 3H, H–40), 1.94–1.80 (m, 2H, H–11a + H–11b), 1.77–1.66 (m, 4H, H–22a + H–22b + H–19a + H–16b), 1.65–1.57 (m, 4H, H–2a + H–2b + H15a + H–1a), 1.57–1.48 (m, 2H, H–9 + H–6a), 1.47–1.30 (m, 3H, H–6b + H–7a + H–21a), 1.27–1.15 (m, 3H, H–7b + H– 21b + H–19b), 1.12 (s, 3H, H–27), 1.09–0.98 (m, 2H, H–15b + H–1b), 0.92 (s, 3H, H–30), 0.91 (s, 3H, H–25), 0.89 (s, 3H, H–29), 0.85 (s, 3H, H–23), 0.84 (s, 3H, H–24), 0.83–0.80 (m, 38

ACCEPTED MANUSCRIPT 1H, H–5), 0.70 (s, 3H, H–26) ppm;

13

C NMR (125 MHz, CDCl3): δ = 177.8 (C28), 171.1

(C39), 170.1 (C37), 170.1 (C35), 144.7 (C13), 132.5 (C32), 121.8 (C12), 119.3 (C33), 81.0 (C3), 55.5 (C5), 52.3 (C38), 52.3 (C31), 51.1 (C34), 48.0 (C17), 47.8 (C9), 46.7 (C19), 43.6 (C18), 42.0 (C14), 41.1 (C36), 39.3 (C8), 38.2 (C1), 37.8 (C4), 37.1 (C10), 34.1 (C21), 33.1 (C29), 32.9 (C7), 30.5 (C22), 30.2 (C20), 28.2 (C15), 28.2 (C23), 26.0 (C27), 24.2 (C30),

RI PT

23.6 (C2), 23.5 (C11), 22.7 (C16), 21.4 (C40), 18.3 (C6), 17.0 (C26), 16.8 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 667.3 ([M+H]+, 100), 689.5 ([M+Na]+, 24), 1355.3 ([2M+Na]+, 60); analysis calculated for C40H62N2O6 (666.93): C 72.04, H 9.37, N 4.20; found: C 71.92, H

4.3.30

(3β)

SC

9.53, N 4.01.

3-Hydroxy-methyl-2-(2-(N-(furan-3-ylmethyl)-olean-12-en-28-carboxamido)

acetamido)acetate (5b)

M AN U

Compound 5b was prepared according to GP A by reaction of OA (250 mg, 0.55 mmol) with furfurylamine, (60 µL, 0.68 mmol), methyl isocyanoacetate (50 µL, 0.55 mmol) and paraformaldehyde (20 mg, 0.67 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 6:4) provided compound 5b (140 mg, 40 %) as a colorless solid; RF = 0.35 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 105–108 °C; RF = 0.35 (silica

TE D

gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); [α]D = +11.76° (c = 0.39, CHCl3); IR (KBr): v = 3425s, 2946vs, 2874m, 1752s, 1684s, 1624s, 1540m, 1438s, 1378s, 1206s, 1178s, 1076m, 1012m, 734m cm–1; 1H NMR (400 MHz, CDCl3): δ = 7.35 (dd, J = 1.5, 0.5 Hz 1H, H–33), 6.67 (dd, J = 5.5, 5.5 Hz, 1H, NH), 6.32 (dd, J = 3.2, 1.9 Hz, 1H, H–34),

EP

6.27 (d, J = 3.2 Hz, 1H, H–35), 5.24 (dd, J = 3.6, 3.6 Hz, 1H, H–12), 4.89 (d, J = 16.1 Hz, 1H, H–31a), 4.69 (d, J = 16.1 Hz, 1H, H–31b), 4.16 (d, J = 15.6 Hz, 1H, H–36a), 4.00 (dd, J = 18.2, 5.5 Hz, 1H, H–38a), 3.91 (dd, J = 18.2, 5.5 Hz, 1H, H–38b), 3.80–3.65 (m, 1H, H–

AC C

36b), 3.72 (s, 3H, H–40), 3.19 (dd, J = 11.1, 4.7 Hz, 1H, H–3), 3.06 (dd, J = 14.9, 4.0 Hz, 1H, H–18), 2.16 (ddd, J = 14.3, 14.3, 3.2 Hz, 1H, H–16a), 1.98–1.75 (m, 5H, H–11a + H–11b + H–16b + H–22a + H–22b), 1.76–1.47 (m, 7H, H–19a + H–9 + H–1a + H–15a + H–2a + H–2b + H–6a), 1.47–1.29 (m, 3H, H–6b + H–21a + H–7a), 1.28–1.14 (m, 3H, H–19b + H–21b + H– 7b), 1.13 (s, 3H, H–27), 1.05 (ddd, J = 13.9, 3.3, 3.3 Hz, 1H, H–15b), 0.97 (s, 3H, H–23), 0.96–0.92 (m, 1H, H–1b), 0.93 (s, 3H, H–30), 0.90 (s, 3H, H–29), 0.89 (s, 3H, H–25), 0.77 (s, 3H, H–24), 0.76–0.69 (m, 1H, H–5), 0.69 (s, 3H, H–26) ppm; 13C NMR (100 MHz, CDCl3): δ = 177.7 (C28), 170.1 (C39), 169.7 (C37), 149.9 (C32), 144.8 (C13), 142.7 (C33), 121.8 (C12), 110.6 (C34), 109.3 (C35), 79.1 (C3), 55.4 (C5), 52.4 (C40), 51.3 (C36), 48.2 (C17), 47.9 (C9), 46.8 (C19), 46.3 (C31), 43.7 (C18), 42.0 (C14), 41.2 (C38), 39.3 (C8), 38.9 (C4), 39

ACCEPTED MANUSCRIPT 38.6 (C1), 37.2 (C10), 34.2 (C21), 33.1 (C29), 32.9 (C7), 30.5 (C22), 30.4 (C20), 28.2 (C23), 28.2 (C15), 27.3 (C2), 26.0 (C27), 24.2 (C30), 23.5 (C11), 23.1 (C16), 18.5 (C6), 17.0 (C26), 15.7 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 665.3 ([M+H]+, 75), 687.5 ([M+Na]+, 15 %), 1016.9 ([3M+K+H]2+, 5), 1329.1 ([2M+H]+, 15), 1346.3 ([2M+NH4]+, 25), 1351.3 ([2M+Na]+, 100); analysis calculated for C40H60N2O6 (664.91): C 72.25, H 9.10, N 4.21;

RI PT

found: 71.96, H 9.20, N 4.07.

4.3.31 (2α,3β) 2,3-Diacetoxy-N-(2-(tert-butylamino)-2-oxoethyl)-N-(prop-2-enyl)-olean-12en-28-carboxamide (6a)

SC

Compound 6a was prepared according to GP A by reaction of acetylated MA (200 mg, 0.36 mmol) with allylamine (40 µL, 0.53 mmol), tert–butyl isocyanide (42 µL, 0.37 mmol) and paraformaldehyde (11 mg, 0.37 mmol). Column chromatography (silica gel, n-hexane/ethyl

M AN U

acetate, 7:3) provided 6a (153 mg, 55 %) as a colorless solid; RF = 0.73 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 129–133 °C; [α]D = –43.57° (c = 0.35, CHCl3); IR (KBr): v = 3442vs, 3432vs, 2966m, 2948m, 2878w, 1744s, 1680m, 1664m, 1638m, 1540w, 1456w, 1394m, 1366m, 1254s, 1232s, 1182w, 1044m, 1034m, 1006w cm–1; 1H NMR (400 MHz, CDCl3): δ = 6.15 (s, 1H, NH), 5.75 (ddd, J = 16.4, 11.0, 5.8 Hz,

TE D

1H, H–36), 5.29–5.19 (m, 3H, H–12 + H–37), 5.09 (ddd, J = 11.4, 10.5, 4.5 Hz, 1H, H–2), 4.73 (d, J = 10.3 Hz, 1H, H–3), 4.33 (d, J = 14.9 Hz, 1H, H–35a), 4.18 (d, J = 14.0 Hz, 1H, H–38a), 3.98 (dd, J = 16.0, 4.2 Hz, 1H, H–35b), 3.47 (br s, 1H, H–38b), 3.08 (dd, J = 11.6, 2.7 Hz, 1H, H–18), 2.11 (ddd, J = 14.2, 14.2, 2.9 Hz, 1H, H–16a), 2.04 (s, 3H, H–34), 2.06–

EP

1.77 (m, 3H, H–1a + H–11a + H–11b), 1.96 (s, 3H, H–32), 1.76–1.56 (m, 6H, H–16b + H–15a + H–22a + H–22b + H–19a + H–9), 1.56–1.49 (m, 1H, H–6a), 1.49–1.33 (m, 3H, H–6b + H– 7a + H–21a), 1.30 (s, 9H, H–41), 1.30–1.14 (m, 3H, H–7b + H–21b + H–19b), 1.12 (s, 3H,

AC C

H–27), 1.04 (s, 3H, H–25), 1.10–0.98 (m, 2H, H–15b + H–1b), 0.99–0.93 (m, 1H, H–5), 0.92 (s, 3H, H–30), 0.90 (s, 6H, H–24 + H–29), 0.88 (s, 3H, H–23), 0.71 (s, 3H, H–26) ppm; 13C NMR (100 MHz, CDCl3): δ = 177.4 (C28), 171.0 (C33), 170.6 (C31), 168.9 (C39), 144.8 (C13), 132.5 (36), 121.5 (C12), 119.1 (C37), 80.8 (C3), 70.2 (C2), 55.1 (C5), 52.2 (C38), 52.0 (C35), 51.2 (C40), 47.9 (C17), 47.8 (C9), 46.7 (C19), 44.0 (C1), 43.4 (C18), 42.1 (C14), 39.5 (C4), 39.3 (C8), 38.4 (C10), 34.2 (C21), 33.0 (C29), 32.7 (C7), 30.5 (C22), 30.3 (C20), 28.9 (C41), 28.6 (C23), 28.3 (C15), 25.9 (C27), 24.1 (C30), 23.6 (C11), 22.7 (C16), 21.3 (C32), 21.0 (C34), 18.4 (C6), 17.8 (C24), 17.2 (C26), 16.6 (C25) ppm; MS (ESI): m/z (%) = 709.3 ([M+H]+, 100), 731.5 ([M+Na]+, 20), 1418.2 ([2M+H]+, 40), 1440.6 ([2M+Na]+, 50); analysis calculated for C43H68N2O6 (709.01): C 72.84, H 9.67, N 3.95; found: C 72.56, H 9.94, N 3.56. 40

ACCEPTED MANUSCRIPT 4.3.32 (2α,3β) 2,3-Diacetoxy-N-(2-(tert-butylamino)-2-oxoethyl)-N-(furan-2-ylmethyl)-olean12-en-28-carboxamide (6b) Compound 6b was prepared according to GP A by reaction of acetylated MA (200 mg, 0.36 mmol) with furfurylamine (50 µL, 0.57 mmol), tert–butyl isocyanide (42 µL, 0.37 mmol) and

RI PT

paraformaldehyde (11 mg, 0.37 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 7:3) gave 6b (173 mg, 64 %) as a colorless solid; RF = 0.75 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 135–138 °C; [α]D = –21.27° (c = 0.32, CHCl3); IR (KBr): v = 3428vs, 2948s, 2878w, 2868w, 1744vs, 1678m, 1636s, 1540m, 1534m, 1

SC

1456m, 1394m, 1368s, 1254vs, 1232s, 1178m, 1162m, 1076w, 1044m, 1034m, 1012m cm–1; H NMR (400 MHz, CDCl3): δ = 7.35 (dd, J = 1.7, 0.6 Hz, 1H, H–37), 6.33 (dd, J = 3.2, 1.9

Hz, 1H, H–38), 6.27 (d, J = 3.1 Hz, 1H, H–39), 5.92 (s, 1H, NH), 5.25 (dd, J = 3.2, 3.2 Hz,

M AN U

1H, H–12), 5.09 (ddd, J = 11.1, 10.4, 4.5 Hz, 1H, H–2), 4.80–4.75 (m, 1H, H–35a), 4.73 (d, J = 10.3 Hz, 1H, H–3), 4.65 (d, J = 15.8 Hz, 1H, H–35b), 4.17 (d, J = 15.3 Hz, 1H, H–40a), 3.61 (d, J = 14.4 Hz, 1H, H–40b), 3.10 (dd, J = 14.1, 2.9 Hz, 1H, H–18), 2.14 (ddd, J = 15.1, 15.1, 3.4 Hz, 1H, H–16a), 2.04 (s, 3H, H–34), 2.04–1.92 (m, 1H, H–1a), 1.97 (s, 3H, H–32), 1.88–1.82 (m, 2H, H–11a + H–11b), 1.82–1.75 (m, 2H, H–22a + H–16b), 1.75–1.64 (m, 2H,

TE D

H–22b + H–16b), 1.64–1.57 (m, 2H, H–9 + H–15a), 1.56–1.50 (m, 1H, H–6a), 1.49–1.31 (m, 3H, H–6b + H–21a + H–7a), 1.28 (s, 9H, H–43), 1.28–1.22 (m, 2H, H–21b + H–7b), 1.21– 1.13 (m, 1H, H–19b), 1.12 (s, 3H, H–27), 1.10–0.99 (m, 2H, H–15b + H–1b), 1.04 (s, 3H, H– 25), 0.99–0.94 (m, 1H, H–5), 0.93 (s, 3H, H–30), 0.90 (s, 6H, H–24 + H–29), 0.89 (s, 3H, H– 13

C NMR (100 MHz, CDCl3): δ = 177.4 (C28), 171.0 (C33),

EP

23), 0.70 (s, 3H, H–26) ppm;

170.7 (C31), 168.4 (C41), 150.2 (C36), 144.9 (C13), 142.5 (C37), 121.4 (C12) , 110.7 (C38), 109.4 (C39), 80.8 (C3), 70.2 (C2), 55.1 (C5), 52.7 (C40), 51.2 (C42), 48.2 (C9), 47.8 (C17),

AC C

46.8 (C19), 46.3 (C35), 44.0 (C1), 43.6 (C18), 42.1 (C14), 39.5 (C4), 39.3 (C8), 38.4 (C10), 34.2 (C21), 33.0 (C29), 32.8 (C7), 30.5 (C22), 30.4 (C20), 28.8 (C43), 28.6 (C23), 28.3 (C15), 25.9 (C27), 24.1 (C30), 23.6 (C11), 23.0 (C16), 21.3 (C32), 21.0 (C34), 18.4 (C6), 17.8 (C24), 17.2 (C26), 16.6 (C25) ppm; MS (ESI): m/z (%) = 749.3 ([M+H]+, 100), 771.5 ([M+Na]+, 20), 1143.0 ([3M+K+H]2+, 5), 1497.3 ([2M+H]+, 35), 1519.4 ([2M+Na]+, 80); analysis calculated for C45H68N2O7 (749.03): C 72.16, H 9.15, N 3.74; found: C 72.00, H 9.23, N 3.46.

4.3.33 (2α,3β) 2,3-Diacetoxy-N-(2-benzylamino-2-oxoethyl)-N-(furan-2-ylmethyl)-olean-12en-28-carboxamide (7a) 41

ACCEPTED MANUSCRIPT Compound 7a was prepared according to GP A by reaction of acetylated MA (200 mg, 0.36 mmol) with furfurylamine (50 µL, 0.57 mmol), benzyl isocyanide (45 µL, 0.37 mmol) and paraformaldehyde (11 mg, 0.37 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 6:4) gave 7a (126 mg, 45 %) as a colorless solid; mp = 137–140 °C; RF = 0.59 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); [α]D = –12.73° (c = 0.32,

RI PT

CHCl3); IR (KBr): v = 3446vs, 2946m, 2878w, 2866w, 1740m, 1646m, 1636m, 1540w, 1472w, 1456w, 1436w, 1394w, 1370m, 1254m, 1178w, 1162w, 1076w, 1042w, 1032w cm–1; 1

H NMR (500 MHz, cdcl3): δ = 7.31–7.26 (m, 3H, H–37 + H–45), 7.26–7.19 (m, 3H, H–46 +

H–44), 6.59 (dd, J = 5.7, 5.7 Hz, 1H, NH), 6.31 (dd, J = 3.2, 1.9 Hz, 1H, H–38), 6.27 (d, J =

SC

3.1 Hz, 1H, H–39), 5.21 (dd, J = 3.0, 3.0 Hz, 1H, H–12), 5.08 (ddd, J = 11.3, 11.3, 4.7 Hz, 1H, H–2), 4.83 (d, J = 15.2 Hz, 1H, H–35a), 4.74–4.68 (m, 2H, H–35b + H–3), 4.41 (dd, J = 14.8, 6.2 Hz, 1H, H–42a), 4.28 (dd, J = 14.8, 5.2 Hz, 1H, H–42b), 4.16 (d, J = 15.3 Hz, 1H,

M AN U

H–40a), 3.76 (br s, 1H, H–40b), 3.03 (dd, J = 10.9, 3.1 Hz, 1H, H–18), 2.13 (dd, J = 15.0, 15.0, 2.8 Hz, 1H, H–16a), 2.04 (s, 3H, H–34), 2.01–1.97 (m, 1H, 1a), 1.96 (s, 3H, H–32), 1.95–1.86 (m, 1H, H–11a), 1.86–1.78 (m, 2H, H–11b + H–16b), 1.78–1.71 (m, 1H, H–22a), 1.71–1.47 (m, 5H, H–22b + H–15a + H–19a + H–9 + H–6a), 1.40–1.28 (m, 3H, H–6a + H– 21a + H–7a), 1.27–1.12 (m, 3H, H–19b + H–21b + H–7b), 1.10 (s, 3H, H–27), 1.09–1.02 (m,

TE D

2H, H–1b + H–15b), 1.01 (s, 3H, H–25), 0.99–0.90 (m, 1H, H–5), 0.89 (s, 6H, H–24 + H–29), 0.89 (s, 3H, H–30), 0.88 (s, 3H, H–23), 0.64 (s, 3H, H–26). ppm;

13

C NMR (125 MHz,

CDCl3): δ = 177.6 (C28), 171.0 (C33), 170.6 (C31), 169.3 (C41), 149.8 (C36), 144.9 (C13), 142.7 (C37), 138.2 (C43), 128.7 (C45), 127.8 (C46), 127.5 (C44), 121.4 (C12), 110.6 (C38),

EP

109.4 (C39), 80.8 (C3), 70.2 (C2), 55.1 (C5), 51.9 (C40), 48.2 (C17), 47.7 (C9), 46.7 (C19), 46.4 (C35), 44.0 (C1), 43.6 (C18), 43.5 (C42), 42.0 (C14), 39.5 (C4), 39.3 (C8), 38.3 (C10), 34.2 (C21), 33.0 (C29), 32.7 (C7), 30.5 (C22), 30.4 (C20), 28.5 (C23), 28.1 (C15), 25.9

AC C

(C27), 24.1 (C30), 23.6 (C11), 23.0 (C16), 21.3 (C32), 21.0 (C34), 18.4 (C6), 17.7 (C24), 17.0 (C26), 16.6 (C25) ppm; MS (ESI): m/z (%) = 783.3 ([M+H]+, 100). 805.5 ([M+Na]+, 20), 837.3 ([M+Na+MeOH]+, 10), 1566.1 ([2M+H]+, 20), 1588.3 ([2M+Na]+, 25) ;analysis calculated for C48H66N2O7 (783.05): C 73.62, H 8.50, N 3.58; found: C 73.44, H 8.69, N 3.41. 4.3.34

(2α,3β)

2,3-Diacetoxy-N-(2-benzylamino-2-oxoethyl)-N-benzyl-olean-12-en-28-

carboxamide (7b) Compound 7b was prepared according to GP A by reaction of acetylated MA (200 mg, 0.36 mmol) with benzylamine (62 µL, 0.57 mmol), benzyl isocyanide (45 µL, 0.37 mmol) and paraformaldehyde (11 mg, 0.37 mmol). Column chromatography (silica gel, n-hexane/ethyl 42

ACCEPTED MANUSCRIPT acetate, 7:3) gave 7b (107 mg, 38 %) as a colorless solid; RF = 0.65 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 143–144 °C; [α]D = +10.34° (c = 0.30, CHCl3); IR (KBr): v = 3440s, 2948s, 2878m, 2866m, 1744vs, 1684m, 1636m, 1540m, 1534m, 1496w, 1454m, 1432m, 1394m, 1368s, 1252vs, 1234s, 1180m, 1042m, 1030m, 698m cm–1; 1H NMR (400 MHz, CDCl3): δ = 7.35–7.17 (m, 10H, H–37 + H–38 + H–39 + H–44 + H–45 +

RI PT

H–46), 6.53 (s, 1H, NH), 5.15 (br s, 1H, H–12), 5.09 (ddd, J = 11.3, 11.3, 4.7 Hz, 1H, H–2), 5.00–4.80 (m, 2H, H–35a + H–35b), 4.73 (d, J = 10.3 Hz, 1H, H–3), 4.43 (dd, J = 14.7, 6.2 Hz, 1H, H–42a), 4.28 (dd, J = 14.8, 5.1 Hz, 1H, H–42b), 4.03 (d, J = 13.6 Hz, 1H, H–40a), 3.80 (br s, 1H, H–40b), 3.00 (dd, J = 11.5, 2.8 Hz, 1H, H–18), 2.14–2.02 (m, 1H, H–16a),

SC

2.05 (s, 3H, H–34), 2.01–1.77 (m, 4H, H–11a + H–11b + H–16b + H–1a), 1.97 (s, 3H, H–32), 1.77–1.56 (m, 5H, H–15a + H–9 + H–19a + H–22a + H–22b), 1.56–1.48 (m, 1H, H–6a), 1.46–1.38 (m, 2H, H–6b + H–7a), 1.38–1.27 (m, 1H, H–21a), 1.26–1.20 (m, 1H, H–7b), 1.20–

M AN U

1.09 (m, 2H, H–19b + H–21b), 1.09 (s, 3H, H–27), 1.09–0.99 (m, 1H, H–1b), 1.03 (s, 3H, H– 25), 1.00–0.93 (m, 2H, H–5 + H–15b), 0.91 (s, 3H, H–24), 0.89 (s, 3H, H–23), 0.88 (s, 6H, H–30 + H–29), 0.74 (s, 3H, H–26) ppm;

13

C NMR (100 MHz, CDCl3): δ = 178.2 (C28),

170.9 (C33), 170.6 (C31), 169.3 (C41), 145.0 (C13), 138.2 (C43), 136.4 (C36), 128.9 (C45), 128.7 (C38), 127.9 (C46), 127.7 (C39), 127.5 (C44), 127.3 (C37), 121.3 (C12), 80.8 (C3),

TE D

70.2 (C2), 55.1 (C5), 53.1 (C35), 51.7 (C40), 48.3 (C17), 47.8 (C9), 46.9 (C19), 44.0 (C1), 43.7 (C18), 43.5 (C42), 42.1 (C14), 39.5 (C4), 39.4 (C8), 38.3 (C10), 34.2 (C21), 33.0 (C29), 32.7 (C7), 30.5 (C22), 30.5 (C12), 28.6 (C23), 28.2 (C15), 25.9 (C27), 24.2 (C30), 23.6 (C11), 23.2 (C16), 21.3 (C32), 21.0 (C34), 18.4 (C6), 17.8 (C24), 17.3 (C26), 16.6 (C25)

EP

ppm; MS (ESI): m/z (%) = 793.3 ([M+H]+, 90), 815.5 ([M+Na]+, 40), 1212.3 ([3M+2Na]2+, 5), 1585.2 ([2M+H]+, 50), 1607.5 ([2M+Na]+, 100); analysis calculated for C50H68N2O6

AC C

(793.09): C 75.72, H 8.64, N 3.53; found: C 75.51, H 8.92, N 3.44.

4.3.35 (3β) 3-Acetoxy-(2-(tert-butyl)amino-2-oxoethyl)-olean-12-en-28-oate (8) Compound 8 was prepared according to GP D by reaction of Ac-OA (200 mg, 0.40 mmol), paraformaldehyde (12 mg, 0.40 mmol) and tert–butyl isocyanide (45 µL, 0.40 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 8:2) gave 8 (220 mg, 90 %) as a colorless solid; RF = 0.69 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 245–247 °C; [α]D = +38.22° (c = 0.36, CHCl3); IR (KBr): v = 3432s, 2950s, 2936s, 2876m, 2862m, 1732vs, 1686s, 1636w, 1522m, 1454m, 1438w, 1390m, 1368m, 1248vs, 1172m, 1164m, 1120m, 1046w, 1038m, 1028m cm–1; 1H NMR (500 MHz, CDCl3): δ = 5.94 (s, 1H, NH), 5.29 (dd, J = 3.5, 3.5 Hz, 1H, H–12), 4.51–4.46 (m, 1H, H–3), 4.39 (s, 2H, H– 43

ACCEPTED MANUSCRIPT 33a + H–33b),,.82 (dd, J = 13.7, 4.2 Hz, 1H, H–18), 2.08–2.00 (m, 1H, H–16a), 2.04 (s, 3H, H–32), 1.88 (dd, J = 8.9, 3.5 Hz, 2H, H–11a + H–11b), 1.73 (ddd, J = 13.8, 13.8, 4.4 Hz, 1H, H–22a), 1.69–1.50 (m, 8H, H–16b + H–2a + H–2b + H–6a + H–15a + H–22b + H–1a + H– 19a), 1.48–1.38 (m, 2H, H–7a + H–21a), 1.38 (s, 9H, H–36), 1.30–1.15 (m, 3H, H–7b + H– 21b + H–19b), 1.14 (s, 3H, H–27), 1.12–0.99 (m, 2H, H–15b + H–1b), 0.94 (s, 3H, H–30),

RI PT

0.92 (s, 6H, H–25 + H–29), 0.86 (s, 3H, H–23), 0.85 (s, 3H, H–24), 0.84–0.79 (m, 1H, H–5), 0.68 (s, 3H, H–26) ppm; 13C NMR (125 MHz, CDCl3): δ = 176.2 (C28), 171.1 (C31), 166.6 (C34), 144.1 (C13), 122.6 (C12), 81.0 (C3), 63.4 (C33), 55.5 (C5), 51.4 (C35), 47.6 (C9), 47.0 (C17), 46.0 (C19), 41.9 (C14), 41.7 (C18), 39.5 (C8), 38.2 (C1), 37.8 (C4), 37.1 (C10),

SC

33.9 (C21), 33.1 (C29), 32.7 (C7), 32.6 (C22), 30.8 (C20), 28.9 (C36), 28.2 (C23), 27.7 (C15), 26.1 (C27), 23.7 (C30), 23.6 (C2), 23.5 (C11), 23.5 (C16), 21.4 (C32), 18.3 (C6), 17.0 (C26), 16.8 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 552.3 ([M+H-HOAc]+, 10), 612.1

M AN U

([M+H]+, 45), 634.4 ([M+Na]+, 10), 1223.4 ([2M+H]+, 100), 1245.4 ([2M+Na]+, 40); analysis calculated for C38H61NO5 (611.90): C 74.59, H 10.05, N 2.29; found: 74.35, N 10.19, N 2.07. 4.3.36 (3β) 3-Acetoxy-(2-benzylamino-2-oxoethyl)-olean-12-en-28-oate (9) Compound 9 was prepared according to GP D by reaction of Ac-OA (200 mg, 0.40 mmol),

TE D

paraformaldehyde (12 mg, 0.40 mmol) and benzyl isocyanide (50 µL, 0.41 mmol). Column chromatography (silica gel, n-hexane/ethyl acetate, 8:2) gave 9 (170 mg, 66 %) as a colorless solid; RF = 0.80 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 139–140 °C; [α]D = +35.40° (c = 0.33, CHCl3); IR (KBr): v = 3442s, 2948s, 2878m, 1734s,

EP

1684m, 1636m, 1540w, 1456m, 1384m, 1366m, 1247s, 1158m, 1122m, 1028w cm–1; 1H NMR (400 MHz, CDCl3): δ = 7.35–7.21 (m, 5H, H–37 + H–38 + H–39), 6.44 (dd, J = 5.5, 5.5 Hz, 1H, NH), 4.99 (dd, J = 3.4, 3.4 Hz, 1H, H–12), 4.65 (d, J = 15.6 Hz, 1H, H–33a), 4.52 (dd, J

AC C

= 14.7, 6.0 Hz, 1H, H–35a), 4.46–4.36 (m, 3H, H–3 + H–33b + H–35b), 2.73 (dd, J = 13.6, 4.1 Hz, 1H, H–18), 2.01 (s, 3H, H–32), 1.98–1.91 (m, 1H, H–16a), 1.73–1.55 (m, 8H, H–16b + H–11a + H–11b + H–2a + H–2b + H–15a + H–19a + H–22a), 1.55–1.43 (m, 4H, H–1a + H–6a + H–22b + H–9), 1.43–1.23 (m, 3H, H–6b + H–7a + H–21a), 1.23–1.08 (m, 3H, H–7b + H–21b + H–19b), 1.06 (s, 3H, H–27), 1.05–0.90 (m, 2H, H–1b + H–15b), 0.86 (s, 3H, H–25), 0.85 (s, 3H, H–29), 0.82 (s, 3H, H–30), 0.81 (s, 3H, H–23), 0.80 (s, 3H, H–24), 0.79–0.74 (m, 1H, H–5), 0.59 (s, 3H, H–26) ppm;

13

C NMR (100 MHz, CDCl3): δ = 176.2 (C28), 171.1

(C31), 167.4 (C34), 144.6 (C13), 137.7 (C36), 129.0 (C38), 127.9 (C39), 127.8 (C37), 122.4 (C12), 80.9 (C3), 63.2 (C33), 55.3 (C5), 47.5 (C9), 47.2 (C17), 45.8 (C19), 43.4 (C35), 41.9 (C14), 41.7 (C18), 39.4 (C8), 38.2 (C1), 37.8 (C4), 37.0 (C10), 33.8 (C21), 33.1 (C29), 32.6 44

ACCEPTED MANUSCRIPT (C22), 32.6 (C7), 30.8 (C20), 28.2 (C23), 27.6 (C15), 26.0 (C27), 23.7 (C30), 23.6 (C2), 23.4 (C11), 23.4 (C16), 21.4 (C32), 18.3 (C6), 17.0 (C26), 16.8 (C24), 15.5 (C25) ppm; MS (ESI): m/z (%) = 586.3 ([M+H-HOAc]+, 10), 646.1 ([M+H]+, 80), 668.4 ([M+Na]+, 50), 988.3 ([3M+K+H]2+, 10), 1291.4 ([2M+H]+, 100), 1313.3 ([2M+Na]+, 98); analysis calculated for

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C41H59NO5 (645.91): C 76.24, H 9.21, N 2.17; found: C 75.96, H 9.37, N 2.03. 4.3.37 (2α,3β) 2,3-Diacetoxy-(2-benzylamino-2-oxoethyl)-olean-12-en-28-oate (10)

Compound 10 was prepared according to GP D by reaction of Ac-MA (200 mg, 0.35 mmol), paraformaldehyde (11 mg, 0.37 mmol) and benzyl isocyanide (42 µL, 0.35 mmol). Column

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chromatography (silica gel, n-hexane/ethyl acetate/chloroform, 8:2:10) gave 10 (143 mg, 58 %) as a colorless solid; RF = 0.73 (silica gel, toluene/ethyl acetate/n–heptane/formic acid, 80:30:10:3); mp = 102–105 °C; [α]D = +4.54° (c = 0.33, CHCl3); IR (KBr): v = 3442s, 2946s,

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2880w, 2866w, 1742vs, 1684m, 1534m, 1456m, 1434w, 1368m, 1245vs, 1234s, 1158m, 1124w, 1042m, 1032m cm–1; 1H NMR (500 MHz, CDCl3): δ = 7.38–7.27 (m, 5H, H–39 + H– 40 + H–41), 6.44 (dd, J = 5.7, 5.7 Hz, 1H, NH), 5.07 (ddd, J = 11.4, 11.4, 4.7 Hz, 1H, H–2), 5.02 (dd, J = 3.4, 3.4 Hz, 1H, H–12), 4.74–4.65 (m, 2H, H–3 + H–35a), 4.54 (dd, J = 14.7, 6.1 Hz, 1H, H–37a), 4.47–4.42 (m, 2H, H–35b + H–37b), 2.77 (dd, J = 13.5, 3.8 Hz, 1H, H–18),

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2.05 (s, 3H, H–34), 1.98 (s, 3H, H–32), 1.98–1.91 (m, 2H, H–16a + H–1a), 1.72–1.59 (m, 5H, H–19a + H–22a + H–11a + H–11b + H–16b), 1.59–1.49 (m, 4H, H–6a + H–15a + H–22b + H–9), 1.48–1.29 (m, 3H, H–6b + H–21a + H–7a), 1.28–1.10 (m, 2H, H–21b + H–7b), 1.09 (s, 3H, H–27), 1.07–0.96 (m, 2H, H–15b + H–1b), 0.93 (s, 3H, H–25), 0.93–0.90 (m, 1H, H–5),

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0.90 (s, 6H, H–24 + H–29), 0.89 (s, 3H, H–30), 0.88 (s, 3H, H–23), 0.60 (s, 3H, H–26) ppm; 13

C NMR (125 MHz, CDCl3): δ = 176.2 (C28), 171.0 (C33), 170.7 (C31), 167.4 (C36), 144.7

(C13), 137.6 (C38), 129.0 (C40), 128.0 (C41), 127.8 (C42), 122.1 (C12), 80.6 (C3), 70.1

AC C

(C2), 63.2 (C35), 54.9 (C5), 47.5 (C9), 47.1 (C17), 45.8 (C19), 43.9 (C1), 43.4 (C37), 41.9 (C14), 41.6 (C18), 39.4 (C4), 39.4 (C8), 38.2 (C10), 33.8 (C21), 33.1 (C29), 32.5 (C7), 32.4 (C22), 30.8 (C20), 28.5 (C23), 27.5 (C15), 26.0 (C27), 23.7 (C30), 23.4 (C11), 23.3 (C16), 21.3 (C32), 21.1 (C34), 18.3 (C6), 17.7 (C24), 16.9 (C26), 16.5 (C25) ppm; MS (ESI): m/z (%) = 704.1 ([M+H]+, 50), 726.5 ([M+Na]+, 35), 1078.0 ([3M+2Na]2+, 10), 1407.3 ([2M+H]+, 70), 1429.3 ([2M+Na]+, 100); analysis calculated for C43H61NO7 (703.95): C 73.37, H 8.73, N 1.99; found: C 73.09, H 8.87, N 1.65.

Acknowledgments

45

ACCEPTED MANUSCRIPT We like to thank Dr. R. Kluge for the measurement of the ESI-MS spectra, Dr. D. Ströhl and his team for the NMR spectra, V. Simon, BSc, for measuring optical rotations, IR and UV-vis spectra and Mr. D. Gottschalk for support in synthesis. The cell lines were kindly provided by Dr. Th. Müller (Dept. of Haematology/Oncology, Martin-Luther Universität HalleWittenberg). Financial support by ScienceCampus Halle (W13004216 to R. C.) is gratefully

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recognized.

Appendix A. Supplementary Material

Supplementary material associated with this article can be found, in the online version, at

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http://

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ACCEPTED MANUSCRIPT Highlights

* Isocyanide-based multicomponent reactions gave 37 triterpenoid Ugi and Passerini products * Coloroimetric SRB assays showed these products to be cytotoxic. * Several maslinic acid derived Ugi products were cytotoxic in low µM range.

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* These compounds induced programmed cell death in part through an apoptosis pathway.