Synthesis Proposal of Immunosuppressive 9,10-secosteroid A from the

Gorgonian Verrucella Umbraculum

Zhaocong Yuan

1,*

, Hanming Li

and Yining Han

Shenzhen College of International Education, Shenzhen, Guangdong 518043, China

Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA 92697, U.S.A.

Johnathan Academy, Vancouver, British Columbia V6M 2V9, Canada

Keywords: Natural Product, Chemical Synthesis, Diols, Molecular Structure, Aromatic Compounds.

Abstract: The proposal of a total synthesis plan of one of a series of four biologically significant 9,10-secosteroids

recently isolated from Verrucella umbraculum, together with several alternative procedures to the synthesis

plan, using widely available chemicals, is described in this work. Immunosuppressive Verrucellol A is a 9,10-

secosteroid with a bicyclic skeletal framework and an aromatic moiety. Herein, the reported proposal

commences via the synthesis of two of the three structural constituents – the α, β-unsaturated aryl ketone

vinyl, and the olefin side chain. Later, the main bicyclic ring structure is constructed via a Robinson

Annulation reaction, followed by the introduction of the olefin side chain by Grignard reductive addition.

Ultimately, an epoxidation-reductive ring opening would complete the synthesis plan.

1 INTRODUCTION

The polycyclic structures of a series of Verrucella

9,10-secosteroids resemble that of vitamin D

(Maestro, Molnár, Carlberg 2019), with the two

rightmost steroidal saturated rings preserved, and a

cleavage of C-9, C-10 bond present. The C-5, C-6 and

C-7, C-8 olefin functional groups are reduced, and

characteristically, a 3-hydroxy-10-methyl

disubstituted benzene ring is attached to the

fragmented third ring, that is, in a steroidal

framework. Variable saturated and unsaturated

substituents are connected to the five-membered ring

of the bicyclic system in this series of 9,10-

secosteroids, with irregular decorations of hydrogen

and hydroxyl groups at various regions. Specifically,

four 9,10-secosteroids, together with their twelve

derivatives, are isolated utilizing acetone extraction

and repeated column chromatography from the

Gorgonian Verrucella umbraculum collected from

the Yongxing Islands (Li, Sun, Tang, Su, Zheng,

Zhang 2021). Interestingly, in the

immunomodulation assay, the majority of the

secosteroids and their derivatives displayed an

inhibitory effect towards the proliferation of CD4

cells, or commonly known as the T helper cells.

Figure.1: Target Molecule with carbon atoms numbered.

Amongst them, Verrucellol A, which features a

C-22, C-23 unsaturated hydrocarbon side chain and

an 8,9-diol moiety, displayed particular

immunosuppressive efficacy, as compared with its 9-

keto and 15-hydroxy counterparts (Li, Sun, Tang, Su,

Zheng, Zhang 2021). Therefore, the potential of using

Verrucellol A upon treating several autoimmune

diseases has been derived, including Type 1 diabetes

(Haskins, Cooke 2011), and systemic lupus

erythematosus (SLE)(He et al 2016), as both these

612

Yuan, Z., Li, H. and Han, Y.

Synthesis Proposal of Immunosuppressive 9,10-secosteroid A from the Gorgonian Verrucella Umbraculum.

DOI: 10.5220/0011249500003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 612-619

ISBN: 978-989-758-595-1

disorders feature CD4

T cells malfunction. The

existing immunosuppressive pharmaceuticals are

mainly glucocorticoids, which are sometimes prone

to a list of psychological and cognitive adverse

effects (Judd et al 2014,

Correction 2014). This

highlights the potential of using 9,10-Verrucellol A

in replacement of the existing glucocorticoid

treatments (once the drug efficacy and toxicity are

determined with further investigations), as quite

significant structural differences are present between

the two genres of immunomodulatory molecules

(Maestro, Molnár, Carlberg 2019,

Li, Sun, Tang, Su,

Zheng, Zhang 2021). Owing to the

immunosuppressive effects Verrucellol A features

towards the proliferation of CD4

T cells, and its

better performance than its counterparts, and the

existing adverse effects of glucocorticoids

immunomodulatory treatments, we were motivated to

propose a synthesis plan of this molecule. Several

suggestions of reaction conditions are also included

at critical procedures of the synthesis.

2 RETROSYNTHETIC ANALYSIS

Figure 2: Retrosynthesis of 9,10-Verrucellol A.

In the analysis of Verrucella 9,10-Secosteroid’s

structure, so as to devise the retrosynthetic strategy, it

is noticed that if the diol at C-8 and C-9 on molecule

1 can be converted to an α, β-unsaturated ketone, the

structure is very close to a product of Robinson

Annulation. On the other hand, since the alcohol on

the benzene ring can be very reactive to bases, it is

decided to protect it with a methyl group until the last

step of synthesis. For the olefin side chain at the top

right, it can be added to the 5-membered ring via a

Grignard reaction. It is concluded that the alkyl group

should be added after the Robinson Annulation for a

cleaner reaction. Since molecule 6 is commercially

available, the main goal is to synthesize molecule 4

Synthesis Proposal of Immunosuppressive 9,10-secosteroid A from the Gorgonian Verrucella Umbraculum

613

and molecule 5. It is envisioned that molecule 4,

which has an olefin on it, can be assembled through a

Wittig reaction. After that, the Riley oxidation and a

substitution reaction can contribute to synthesize

molecule 2. Considering the fact that an α, β-

unsaturated ketone is quite electrophilic, it is decided

to add the terminal olefin last when constructing

molecule 5. Thus, the synthesis plan is proposed to

start with a benzene derivative 11, then extend the

carbon chain and make a Weinreb amide, which an

addition of Grignard reagent can result in the desired

molecule 5. (Schematic diagram of retrosynthetic

strategy see Figure 2.)

3 FORWARD SYNTHESIS

PROPOSAL

Figure 3: Synthesis of ether-protected aryl constituent 10.

The proposal of the synthesis of Verrucellol A begins

with the regioselective synthesis of ether-protected

aryl constituent 10. Due to the commercial

availability of 2-methyl benzaldehyde 7, this is

chosen as the starting material of the procedure. By

regioselectively sulfonating the meta- position with

respect to the aldehyde, followed by reflux heating

with NaOH and then an acidic workup, the meta-

regioselective hydroxylation of the benzene

derivative can be achieved. In order to prevent the

phenol 9 from further reactions with alkaline

conditions described below, the use of methyl ether

as its protection is incorporated (MeI, K

acetone, reflux) (Greene, Wuts 1999a). This series of

processes (see Figure 3) should give the ether-

protected aryl constituent 10 in quite good yields.

Figure 4: Forward Synthesis of Weinreb Amide 21.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

614

The synthesis of ether-protected aryl ketone vinyl

5, which is vital to the construction of the six-

membered ring of the bicyclic skeleton, commences

with commercially available 4-hydroxy-2-butanone

11. In order to install the carbon chain from C-5 to C-

8 (same labelling as in Verrucellol A molecule) via a

Wittig reaction, it is necessary to substitute the

hydroxyl group of butanone 11 into a PPh

moiety,

making it the Wittig reagent 14. This can be

actualized by ketal protection of the carbonyl

(ethylene glycol, tosylic acid, benzene, reflux)

(Greene, Wuts 1999b), and then bromination utilizing

HBr; the bromine substituted ketal 13 can then be

treated with PPh

, followed by n-butyllithium

deprotonation, which yields the desired phosphonium

ylide 14. The Wittig olefination can thus be

commenced by adding the ether-protected aryl

constituent 10 to ylide 14 in THF (Maryanoff, Reitz

1989), generating the 6,7-cis unsaturated aromatic

ketal 15. This ketal 15 can then be reduced by

hydrogen gas over platinum so as to establish the

saturated C-6, C-7 σ system. Platinum has been

selected as the transition-metal catalyst as it is almost

inactive towards other reactions to olefin than

hydrogenation, such as olefin cis-trans isomerization

(Bond, Wells 1965). The saturated ketal 16 is then

subjected to deprotection at carbonyl to yield the aryl

ketone 17 (PPTs, H

O, acetone, heat) (Greene, Wuts

1999b). As linking the vinyl moiety to the aromatic

ketone 17 via Grignard addition with Weinreb amide

21 is intended, the conversion of aryl ketone 17 into

its carboxylic acid derivative 18 is a sound decision.

It is envisioned that this functional group

interconversion can be realized by a chloroform

oxidation (excess Cl

, NaOH, methyl alcohol, then

HCl workup), for comparing with other halogen

counterparts, hypochlorite facilitates the reaction

kinetics most, raising the yield and simplifying the

separation procedures rather significantly (Fuson,

Bull 1934, VanArendonk, Cupery 1931). The desired

Weinreb amide 21 can be yielded from the carboxylic

acid 18 in a one-pot, transition-metal-free reaction

with N, O-dimethylhydroxylamine 19 (PCl

, toluene

20)(Huang, Hu, Niu, Wang, Xu, Su, Fu 2013). This

method is no longer prone to moisture and air

sensitivity during storage, which may sometimes

become problematic (Niu, Zhang, Huang, Xu, Wang,

Hu 2009). (Schematic diagram see Figure 4.)

Figure.5 Grignard Addition to Weinreb Amide 21 to form molecule 5.

Then, the ether-protected aryl ketone vinyl 5 can

be easily granted by adding the vinyl Grignard 22 to

the Weinreb amide 21 in THF followed by an HCl

workup, as the formation of a stabilized O-Mg-O

chelating intermediate in this process prevents double

addition of vinyl moiety. This is an intermediate that

can be easily removed by an acidic workup to

generate the desired α, β-unsaturated ketone 5

(Nahm, Weinreb 1981). (see Figure 5)

Figure 6: The Synthesis of Grignard Reagent 4.

Synthesis Proposal of Immunosuppressive 9,10-secosteroid A from the Gorgonian Verrucella Umbraculum

615

The synthesis of the olefin side chain (see Figure

6) and its attachment to the main framework could be

initiated by introducing 2,3-dimethylbutanol 23,

which is widely accessible. In an attempt to prepare a

proper ylide for future reactions, a

triphenylphosphine group could substitute the OH

group on the α carbon. A proposed method is to

substitute via HBr as such reactions take place under

relatively milder conditions. The yielded 1-bromo-

2,4-dimethylbutane 24 could subsequently react with

PPh

under strongly basic conditions (for example,

with the presence of n-BuLi), therefore producing the

desired ylide 25. A Schlosser-modified Wittig

reaction with propionaldehyde 26 could then be

conducted to give 5,6-dimethyl-3-heptene 27, which

features the desired E-stereoselectivity (Sondheimer,

Mechoulam 1957, Schlosser, Christmann 1966) with

a subsequent workup. With Schlosser modification,

at a low temperature and basic conditions, the

oxaphosphetane intermediate formed (Vedejs, Marth

1990, Vedejs, Snoble 1973) favors the formation of

E-stereoisomer concomitantly due to the stabilizing

effect of lithium to the ylide and the repulsion

between the bulky moieties.

The olefin side chain could be attached to a

bicyclic ring via a Grignard reaction. To prepare the

Grignard reagent 4, allylic hydroxylation by selenium

dioxide could be applied in an attempt to oxidize the

C-20 and prepare an enol. Such method introduces a

hydroxyl group selectively without any structural

rearrangement, whereas most other methods of

oxidizing allylic carbons do not have this advantage

of regioselectivity and would often prefer reacting

with the tertiary carbon, therefore leading to

undesired products. Studies have shown that it is a

sterically-driven reaction (Stephenson, Speth 1979).

There are two allylic carbons where an addition could

take place—a secondary carbon, C-20, and a tertiary

carbon, C-24. Therefore, the addition of OH group

would, presumably, take place on C-20. To complete

the preparation of the olefin side chain, the OH group

is substituted by Br so as to proceed to prepare the

Grignard reagent 4. With ether as a solvent,

magnesium reacts with 2-bromo-5,6-Dimethyl-3-

heptene 29 to give the final Grignard reagent 4

(Seyferth 1979).

Figure.7: Schlosser Modification Mechanism.

Figure.8: Robinson Annulation and Reductive Ring Opening to reach target molecule.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

616

With molecule 4 and 5 synthesized, it is ready to

assemble the target molecule. The proposed method

commences by executing a Robinson annulation with

molecule 5 and 6 and results in molecule 3 (Li 2009).

There are currently two carbonyls on molecule 3.

Since the α, β-unsaturated ketone is conjugated, its

resonance effect makes it less likely to react with

Grignard reagent 4 than the other ketone (at C-17).

Therefore, the addition of Grignard reagent 4 will be

more likely to happen at C-17. In order to eliminate

the extra OH group (at C-17) formed during Grignard

reaction, the ketone at C-9 is first protected with

ethylene glycol (ethylene glycol, tosylic acid,

benzene, reflux) (Greene, Wuts 1999b); then, the

hydroxy group at C-17 is tosylated to make it a better

leaving group, followed by the use of LiAlH

as a

base to substitute the tosyl group with an H, resulting

in molecule 2. In order to prevent the α, β-unsaturated

ketone from reacting with LiAlH

, ketal (ethylene

glycol, tosylic acid, benzene, reflux) is used to protect

it until OH group at C-17 is eliminated; molecule 32

is then subjected to de-protection (PPTs, H

acetone, heat) (Greene, Wuts 1999b). The next step

of the synthesis is to reduce the ketone to an alcohol

and add an OH to the olefin at α-position. Such

procedure is proposed as it achieves both of them

through a reductive epoxide opening reaction. H

and KOH are used to construct an epoxide at the α-

position, then LiAlH

will be added. Since ketone is

an electron withdrawing group, it makes the olefin at

C-8 more reactive to nucleophilic epoxidation than

the olefin at C-22 with alkyl electron donating groups

around it. LiAlH

first reduces the carbonyl to an

alcohol, then the Al will chelate with both oxygen

atoms; therefore, when the LiAlH

attacks the

epoxide, it is envisioned to attack C-14, subsequently

opening the epoxide ring and forming molecule 34

(Diehl 1937). The last step of the synthesis is to de-

protect the MeO group (BBr

, CH

) on the

benzene ring, which gives us the target molecule

(Greene, Wuts 1999a).

4 EVALUATIONS

The purpose of this proposal is to offer an insight into

the synthesis of a potentially biologically significant

molecule, so experimentations are necessary when

realizing this scheme in laboratories. To further

elaborate the synthesis, efforts could be endeavored

to select a route that features diastereoselectivity of

9,10-Verrucellol A. Nonetheless, we will include

below several suggestions which could be taken into

account if unexpected yields are derived from

particular procedures.

As sometimes, too reactive reagents (such as

strong acids and alkalis, high temperatures and

pressures, repetitive heating and so on) might lead to

undesired functional group interconversions, which is

problematic. One potential manifestation of this

repercussion lies with the regioselective synthesis of

ether-protected aryl constituent 11. Included in the

aforementioned synthesis scheme is the utilization of

the Dow process (sulfonation, NaOH reflux, workup)

with 2-methyl benzaldehyde 20, but from practical

perspectives, the high temperature in alkaline

conditions may degrade the desired aldehyde

functional group.

Figure.9: Pd-catalyzed synthesis of ether-protected aryl constituent 11 (left); with structure of t-BuXPhos ligand elucidated

(right).

Synthesis Proposal of Immunosuppressive 9,10-secosteroid A from the Gorgonian Verrucella Umbraculum

617

Instead, a palladium-catalyzed, microwave-

assisted alkaline hydroxylation of aryl chloride 35

(see Figure 9), with the help of Herrmann’s

Palladacycle (Herrmann, Brossmer, Reisinger,

Riermeier, ÖFele, Beller 1997) and t-BuXPhos

ligand, can be incorporated (Yu, Chen, Huang, Chern

2012). This method has been proved to maintain high

yields even with aldehyde substituents present on the

benzene ring, as milder reaction conditions are

featured. Therefore, the modified reaction cascade

would be as follows: aryl chlorination (liquid Cl

FeCl

), then palladium-catalyzed hydroxylation

(Herrmann’s Palladacycle, t-BuXPhos, Cs

DMF/H

O (9:1), microwave) (Yu, Chen, Huang,

Chern 2012), followed by methyl ether protection

(MeI, K

, acetone) (Greene, Wuts 1999a), giving

the desired ether-protected aryl constituent 11.

It should be noted that in the synthesis proposal,

it has been hypothesized that for molecule 3, the

ketone at C-17 reacts more readily with Grignard

moiety than the α, β-unsaturated ketone at C-9.

Nonetheless, if proved otherwise in experimentations

(by getting more product molecules whose bicyclic

system is linked to the olefin side chain at C-9), a

back-up synthesis route can be adopted: the α, β-

unsaturated ketone at C-9 is first protected (ethylene

glycol, tosylic acid, benzene, reflux) (Greene, Wuts

1999b), followed by reductive addition with Grignard

reagent 4 in THF, which yields molecule 31 directly.

The synthesis can then proceed in the same cascade

as shown in Figure 8.

5 CONCLUSIONS

In summary, because of its biochemical significance

and potential pharmacological efficacy, a synthesis

scheme of 9,10-Verrucellol A has been proposed,

using widely accessible starting materials and a

relatively straightforward synthesis route, which

features constructing the bicyclic skeleton with a

Robinson Annulation, followed by joining the olefin

side chain by a Grignard addition, and approaching

the structure of target molecule with several fine

adjustments. A number of proposed reaction

conditions have also been included herein. This paper

could act as a foundation for further investigations,

such as diastereoselective synthesis of 9,10-

Verrucellol A; in vivo assessments of the target’s

pharmacological efficacy could also be endeavored to

evaluate its practical use.

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