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WO 2007015837 A2 

Antineoplaston (ANP) is a name used by Stanislaw Burzynski for a group of chemical compounds andmixtures for which he claims anti-cancer  activity. These compounds have been sold and administered byBurzynski to cancer patients since 1986; clinical efficacy has not been demonstrated and several fatal sideeffects have occurred.[1] The practice is considered quackery by critics.[2] Burzynski maintains corporate officesand operates a clinic in Houston, Texas where he treats patients with antineoplastons. At a nearby facility inStafford, Texas he conducts research and manufactures the pharmaceutical ingredients used in themedications and other products that he produces.

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Contents

1 Background

2 Treatment with Antineoplastons

3 Proposed mechanisms

4 References

Background

In 1967 Stanislaw Burzynski began investigating the use of antineoplastons after noting significant peptidedeficiencies in the blood of cancer patients as compared with a control group[3]. Burzynski initially derivedantineoplastons from human blood. Since similar peptides had been isolated from urine, in 1970 Burzynskiswitched to urine as a cheaper source of antinoeplastons. Since 1980 he has been reproducing his compoundssynthetically.[4] Since his initial discovery, Burzynski has isolated dozens of peptide fractions from urine, someof which have been reportedly found to be active against cancer with low toxicity.

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The first active peptide fraction identified was called antineoplaston A-10 (3-phenylacetylamino-2,6-piperidinedione). From A-10, antineoplaston AS2-1, a 4:1 mixture of  phenylacetic acid andphenylacetylglutamine, was derived [5]. The active ingredient of antineoplaston A10-I isphenylacetylglutamine [6]. 

Phenylacetic acid is a toxic compound that the body produces during normal metabolism. It is detoxified inthe liver  to phenylacetyl glutamine. The "antineoplaston A-10" compound is an isolation artifact resulting fromheating the urine under acidic conditions. The "antineoplaston AS2-1" mixture is the result of an alkalinehydrolysis of "antineoplaston A-10". All compounds are widely available cheap chemicals.

Treatment with Antineoplastons

For legal reasons Burzynski currently sells his treatments only in the context of clinical trials. Patients receivingcancer treatment with antineoplastons must first qualify for one of the currently available clinical trials. In order to qualify for most of the trials, a patient must have first failed standard treatment for the condition beingtreated, or it must be a condition that is unlikely to respond to currently available therapy and for which nocurative therapy exists. Antineoplastons may be administered intravenously or orally. Patients who respondpositively to initial treatment with intravenous antineoplastons sometimes transition to the oral form.Intravenous antineoplastons are administered continuously with a portable programmable pump that the patientcarries on a shoulder strap in a canvas bag.

Treatment with antineoplastons can be very costly to patients without insurance coverage, exceeding $100,000for the first year of intravenous treatment. Many insurance companies consider antineoplaston therapy to beinvestigational and unproven and will not cover the cost.[7][8] The administered "antineoplastons" are very cheapand widely available chemicals that cost no more than 80 cents per treatment [9]. 

Proposed mechanisms

 Antineoplastons have never shown to be effective in treating human cancer. Independent tests at atthe National Cancer Institute have never been positive.[10] The drug company Sigma-Tau Pharmaceuticalscould not duplicate Burzynski's claims for AS-2.1 and A-10. The Japanese National Cancer Institute hasreported that antineoplastons did not work in their studies. No scientific coauthor of Burzynski publications has

endorsed his use of antineoplastons in cancer patients.[9]

 

Burzynski suggest that antineoplastons A10 and AS2-1 both work by inhibiting oncogenes,promoting apoptosis, and activating tumor suppressor genes [6]. Several other mechanism of action have beenproposed.

One of the factors that allows some cancers to grow out of control is the presence of abnormal enzymes, abyproduct of  DNA methylation. In the presence of these enzymes, the normal life cycle of the cells is disruptedand they replicate continuously. Antineoplastons have been shown in the laboratory to inhibit theseenzymes [11]. 

Recent studies have shown that inhibiting histone deacetylase (HDAC) promotes the activation of  tumor suppressor genes p21 and p53. Phenylacetic acid contained in the AS2-1 mixture has been shown to be a

weak HDAC inhibitor [12]

. 

References

1. ^ (1998) "Burzynski probe finds unflattering picture". NCRHI News 21 (5).2. ^ Goldberg P (1998). "The Antineoplaston Anomaly: How a Drug Was Used for Decades in Thousands

of Patients, With No Safety, Efficacy Data". The Cancer Letter  24 (36):  –.3. ^ Burzynski SR (1986). "Antineoplastons: history of the research (I)". Drugs under experimental and 

clinical research 12 Suppl 1: 1-9. PMID 3527634.4. ^ Ralph Moss (1996), The Cancer Industry ISBN 1881025098

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5. ^ NCI Drug Dictionary, Definitions of antineoplastons A10 and AS2-16. ^ a  b  S.R. Burzynski, The Proposed Mechanism of Antitumor Activity of Antineoplastons (ANPs) in High

Grade Glioma Pathology (HBSG) Integrative Cancer Therapies 2006; 40-477. ^  Aetna Clinical Policy Bulletin, Antineoplaston Therapy and Sodium Phenylbutyrate8. ^ Blue Cross/Blue Shield Medical Policy, Antineoplaston Therapy9. ^ a  b  Saul Green, Stanislaw Burzynski and "Antineoplastons", adapted from a talk at the American

 Association for Clinical Chemistry Symposium, Atlanta, 1997.

10. ^ Burzynski SR (1999). "Efficacy of antineoplastons A10 and AS2-1". Mayo Clin. Proc. 74 (6): 641-2.PMID 10377942.

11. ^ Liau MC, Burzynski SR (1986). "Altered methylation complex isozymes as selective targets for cancer chemotherapy". Drugs under experimental and clinical research 12 Suppl 1: 77-86. PMID3743383.

12. ^ Jung M (2001). "Inhibitors of histone deacetylase as new anticancer agents". Curr. Med.Chem. 8 (12): 1505-11. PMID 11562279.

SYNTHESIS OF 1,4-DI-T-BUTYL-2,5,-DIMETHOXYBENZENE 

Elizabeth Hackett

Organic Chemistry II Lab

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Dr. Jones

10/17/2013

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PURPOSE:  The purpose of this experiment is to investigate the principles of Friedel – Crafts

alkylation reactions through the synthesis of 1,4-di-t-butyl-2,5,-dimethoxybenzene by reacting

1,4-dimethoxybenzene with t-butyl alcohol in the presence of sulfuric acid. 

THEORY  Friedel-Crafts Alkylation reactions are a method of introducing an alkyl substituent

into an aromatic compound through electrophilic aromatic substitution. A catalyst is required for 

the ring to undergo substitution and lose its aromaticity for a short time. The catalysts and co-

reagents involved in the reaction serve to generate the strong electrophilic species needed to

carry out the initial step of the substitution. Most importantly, the existence of substituents on the

 benzene ring prior to reaction affects both the rate of the reaction and orientation of the future

substitution group. Substituents can inductively donate or withdraw electrons through the σ

 bonds of the benzene ring, or donate or withdraw density through resonance with benzene’s π

 bonding system. Substituents are classified as activating or deactivating toward substitution by

either donating electron density or withdrawing electron density from the aromatic system.

Activating groups are those that increase the electron density in the benzene ring, making

it more nucleophilic, thus activating the ring towards electrophilic attack. These electron-

donating groups stabilize the carbocation intermediate, which leads to lower G‡ and faster 

reaction rates. Substituents that activate the benzene ring toward electrophilic attack generally

direct substitution to the ortho and para locations, thus activating groups are ortho-para-

directors. This is because EDG’s make the ortho and para  positions more nucleophilic and

attacking these locations forms a more stable cation than a meta attack. Substituents that have R 

groups or have an atom bonded to the ring that has an unshared pair of electrons are ortho-para

directing.

On the other hand, deactivating groups are those that decrease the electron density and

deactivate the ring towards electrophilic attack. These electron-withdrawing groups destabilize

carbocation intermediate, which lead to higher G‡ and slower rates of reaction. With some

exceptions, such as the halogens, deactivating substituents direct substitution to the meta

location. All meta-directors have a full or partial positive charge on the atom bonded to the

 benzene ring. Because of this, meta directors inductively destabilize the σ complex, thereby

discouraging substitution at sites ortho or para to the substituent. By default, the reactive site is

the meta position, because it is not directly destabilized by the substituent.

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In this experiment, we converted 1,4-dimethoxybenzene into 1,4-di-t-butyl-2,5,-

dimethoxybenzene. This reaction illustrates the Friedel-Crafts alkylation of an activated benzene

molecule with a tertiary alcohol in the presence of sulfuric acid as the Lewis acid catalyst. The

overall reaction is shown below:

The first step of the reaction involves the protonation of tert-butyl alcohol, followed byloss of water. Thus, an electrophilic tert-butyl carbocation is generated through the reaction of 

tert-butyl alcohol with sulfuric acid acting as the dehydrating agent. This reaction is shown

 below:

The advantage of this method for generating the electrophile is that the tertiary butyl

cation is a stable carbocation and will not undergo rearrangement. In the next step, 1,4-

dimethoxybenzene reacts with the carbocation generated from tert-butyl alcohol, and a

trisubstituted product, 1-tert-butyl-2,5-dimethoxybenzene is formed. This involves the addition

of the electrophilic carbocation to the electron-rich aromatic system. Benzene uses two of its π

electrons to react with the electrophile, forming a σ-complex or arenium ion. The σ-complex

subsequently loses a proton to reconstitute the aromatic system, while also regenerating the

catalyst. The arenium ion, intermediate carbocation, is stabilized by resonance, which delocalizes

its charge. This alkylation occurs quite readily because the aromatic ring is activated toward

electrophilic aromatic substitution by the two electron-donating (activating) methoxy groups.

Because the methoxy groups are ortho-para directors, alkylation occurs ortho to the methoxy

group present. This reaction and the three resonance structures represented in this process are

shown below:

Formation of Tert-butyl Carbocation via Dehydration of Protonated Alcohol 

Overall Reaction: C8H10O2 + 2 C4H10O C16H26O2 + 2 H2O

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In Friedal-Crafts reactions, polysubstitution frequently occurs because alkyl groups are

activating and increase the nucleophilicity and reactivity of the ring. Since this trisubsituted

 product contains two activating methoxy groups and one activating tert-butyl group, it

is more reactive in the EAS reaction than 1,4-dimethoxybenzene. Thus, the first alkyl group

activates the ring to the second alkylation. As a result, 1-tert-butyl-2,5-dimethoxybenzene

continues to react with a second equivalent of the tertiary carbocation derived from tert-butyl

alcohol, to generate 1,4-di-tert-butyl-2,5-dimethoxybenzene. The final product is labeled in the

reaction mechanism below:

The 1, 4 positioning of the product was determined by the steric hinderance resulting

from the locations of the methoxy groups. No further substitution reaction occurs because the

two remaining unsubstituted sites on the benzene ring are too sterically hindered.

Electrophilic Attack & Proton Loss 

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PROCEDURE Approximately 6.0g of 1,4-dimethoxybenzene was weighed out and added

into a 125 ml Erlenmeyer flask. To this, 10mL of t-butanol and 20 mL of acetic acid were added,

and the Erlenmeyer flask was placed in an ice bath, cooling the temperature of the mixture to 0-

30C. A thermometer was immersed in the mixture to check and

maintain its temperature. This ice bath was then moved into a

 position under a separatory funnel. The apparatus for this step is

shown in Figure 1. Next, 30 mL of concentrated sulfuric acid was

added to another 50 mL Erlenmeyer flask and was cooled in ice-

water bath. After the sulfuric acid was chilled, it was added to the

separatory funnel. Next, the chilled sulfuric acid was added drop-

wise over a time period of approximately seven minutes into the

Erlenmeyer flask. Occasional stirring was performed during

addition of the sulfuric acid, and the temperature was maintained between 15-200C throughout

the process. The Erlenmeyer flask was then removed from the ice bath, and the mixture was

further maintained between the temperatures 15-200C for an

additional 5 minutes in order to complete the reaction. After this

 period, the flask was chilled once again while the suction

filtration apparatus in figure 2 was constructed. The Erlenmeyer 

flask was then filled with ice, and water was added to until the

flask was full. Next, the flask was poured into the Buchner 

funnel for vacuum filtration, and the product was collected. The

 product was then washed with water, while suctioned, to remove

any residual acids. After this period, the crystals were washed

under suction with three10mL rounds of cold methanol and

were washed and dried. The crystals were then left until the next lab session. The following

week, the crystals were recrystallized by adding approximately 30-35 mL of methanol to the

flask and heating the flask on a hot plate until boiling. The solution was then cooled in an ice

 bath and was filtered using suction filtration. The product was then collected, dried, and

weighed, and the melting point of the product was obtained.

Figure 2 

Figure 1

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TABLE OF R EACTANTS & PRODUCTS 

DATA AND CALCULATIONS 

ACTUAL YIELD: 5.5 G 

THEORETICAL YIELD:  10.86 G, 0.043 MOL 

PERCENT YIELD: 50.6  %

MELTING POINT: TRIAL 1: 99.4 AVG: 99.6 °C

TRIAL 2: 99.8

Limiting Reagent & Theoretical Yield

6.0 g 1,4-dimethoxybenzene (DMOB) ( ) (

) ( ) = 10.86 g product 

10 mL t-Butyl Alcohol (TBA) ) ( ) (

) ( ) (

)= 13.25 g product  

The limiting reagent for this Friedel-Crafts alkylation reaction is 1,4-dimethoxybenzene.

OBSERVATIONS AND R EMARKS During experimentation many notable observations were

made. First, the product obtained directly after addition of slow sulfuric acid addition had a

slightly pinkish color. The flask become noticeable warm upon the addition of sulfuric acid,

signifying this was an exothermic reaction. Also, a yellowish color was observed during

recrystallization, which possibly indicated impurities. Lastly, the resulting product, 1,4-di-t-

 butyl-2,5,-dimethoxybenzene was obtained, which was a white crystalline solid, feathery in

texture. The low percent yield (52%) could be due to the fact that many of the crystals were

stuck in the reaction tube and could not be removed for weighing.

Name StructureMol. Wt.

(g)

Amt

Used

Mol

UsedEq.

b.p/m.p

(°C)

Density

1, 4-

dimethoxybenzene 138.16 6.0 g 0.0434 1.00213

55-59

1.053

g/cm3 

t-butyl alcohol  74.12 10 mL 0.1053 2.0082.8

25-26

0.775

g/mL

Concentrated H2SO

4 98.08 30 mL 0.5628 -290-335

10

1.84

g/mL

1,4-di-t-butyl-2,5,-

dimethoxybenzene250.37 - - -

104-105

336.3 0.924g/cm3 

Solubility data: 1, 4-dimethoxybenzene- Soluble in acetone. Very soluble in ether, benzene.

t-butyl alcohol: Miscible in ester, aromatic and aliphatic hydrocarbons. Soluble in water. Miscible in alcohol andether. 1,4-di-t-butyl-2,5, dimethoxybenzene: very low solubility in water and methanol

Concentrated H2SO

4: Soluble in acetic acid water,proportions in ethanol.