IE20011021U1 - Anti-viral compounds - Google Patents
Anti-viral compoundsInfo
- Publication number
- IE20011021U1 IE20011021U1 IE2001/1021A IE20011021A IE20011021U1 IE 20011021 U1 IE20011021 U1 IE 20011021U1 IE 2001/1021 A IE2001/1021 A IE 2001/1021A IE 20011021 A IE20011021 A IE 20011021A IE 20011021 U1 IE20011021 U1 IE 20011021U1
- Authority
- IE
- Ireland
- Prior art keywords
- formula
- oligomer
- hiv
- hal
- drug
- Prior art date
Links
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- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 101710042981 SHMT1 Proteins 0.000 description 1
- 101710017884 Segment-8 Proteins 0.000 description 1
- 241000713311 Simian immunodeficiency virus Species 0.000 description 1
- 229960001203 Stavudine Drugs 0.000 description 1
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- LSNNMFCWUKXFEE-UHFFFAOYSA-L Sulphite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 229940023080 Viracept Drugs 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 102000016350 Viral Proteins Human genes 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- USDJGQLNFPZEON-UHFFFAOYSA-N [[4,6-bis(hydroxymethylamino)-1,3,5-triazin-2-yl]amino]methanol Chemical compound OCNC1=NC(NCO)=NC(NCO)=N1 USDJGQLNFPZEON-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 231100000494 adverse effect Toxicity 0.000 description 1
- 229920001586 anionic polysaccharide Polymers 0.000 description 1
- 150000004836 anionic polysaccharides Chemical class 0.000 description 1
- 230000000798 anti-retroviral Effects 0.000 description 1
- 229960000070 antineoplastic Monoclonal antibodies Drugs 0.000 description 1
- 229960001716 benzalkonium Drugs 0.000 description 1
- CYDRXTMLKJDRQH-UHFFFAOYSA-N benzyl-dodecyl-dimethylazanium Chemical compound CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 CYDRXTMLKJDRQH-UHFFFAOYSA-N 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000005591 charge neutralization Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 125000002340 chlorooxy group Chemical group ClO[*] 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- WREGKURFCTUGRC-POYBYMJQSA-N ddC Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)CC1 WREGKURFCTUGRC-POYBYMJQSA-N 0.000 description 1
- BXZVVICBKDXVGW-NKWVEPMBSA-N ddIno Chemical compound O1[C@H](CO)CC[C@@H]1N1C(NC=NC2=O)=C2N=C1 BXZVVICBKDXVGW-NKWVEPMBSA-N 0.000 description 1
- 229960005319 delavirdine Drugs 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 201000008286 diarrhea Diseases 0.000 description 1
- 201000009910 diseases by infectious agent Diseases 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 230000002255 enzymatic Effects 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
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- 238000005755 formation reaction Methods 0.000 description 1
- 230000002496 gastric Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 102000034448 gene-regulatory proteins Human genes 0.000 description 1
- 108091006088 gene-regulatory proteins Proteins 0.000 description 1
- 239000001963 growth media Substances 0.000 description 1
- KJUGUADJHNHALS-UHFFFAOYSA-O hydron;2H-tetrazole Chemical compound C1=NN=[NH+]N1 KJUGUADJHNHALS-UHFFFAOYSA-O 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000000977 initiatory Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N iso-propanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 125000006357 methylene carbonyl group Chemical group [H]C([H])([*:1])C([*:2])=O 0.000 description 1
- 229960000060 monoclonal antibodies Drugs 0.000 description 1
- 102000005614 monoclonal antibodies Human genes 0.000 description 1
- 108010045030 monoclonal antibodies Proteins 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
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- 238000006386 neutralization reaction Methods 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
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- 101700002291 ompT Proteins 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 229960001243 orlistat Drugs 0.000 description 1
- 238000003359 percent control normalization Methods 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002250 progressing Effects 0.000 description 1
- 230000001681 protective Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 108010076805 snowdrop lectin Proteins 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002459 sustained Effects 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 231100000925 very toxic Toxicity 0.000 description 1
Description
Anti-viral compounds
Field of the Invention
The present invention relates to compounds having biological activity and to
processes for the preparation thereof. The invention is particularly directed to
compounds having anti-viral, particularly anti-HIV activity.
Background to the Invention
The virus that causes AIDS, the human immunodeficiency virus HIV is believed
to be one of the major threats to human life and health worldwide. Even back in 1988 an
article in Scientific American by J .M. Mann, J. Chin, P. Piot and T. Quinn estimated that
more than a quarter of a million AIDS cases had occurred in the U.S.A. up to then and
that 5-10 million people were infected worldwide. An article in the same magazine ten
years later “Defeating Aids: What will it take? (July 1998 page 62) revealed that
worldwide 40 million people had contracted HIV and almost 12 million had died leaving
over 8 million orphans. During 1997 alone nearly 6 million people acquired HIV and
some 2.3 million perished including 460,000 children.
Although 90% of HIV infected people live in developing countries Well over
90% of money for care and prevention is spent in industrial countries. The very
expensive triple therapy drugs (over US$l0,000-$15,000 per person per year) are well
beyond the reach of individuals in developing countries in sub Saharan Africa and Asia.
In 1999 alone, 300,000 people died in Ethiopia from AIDS far exceeding deaths from
famine (12 April 2000, The Irish Examiner). Up to a quarter of South Africa’s non-
whites currently face death from AIDS in the next ten years (11 May 2000, The Irish
Examiner, by G. Dyer). There is thus a desperate need for cheap, easily made and
efficient anti-HIV agents for the developing world.
The HIV has been studied more intensively than any other virus and we now
have a general picture of how the genes and proteins in the HIV virus particle operate,
although we don’t have a clear understanding of what controls the replication and how it
destroys the human immune system.
main ones are HIV-1 and HIV-2. HIV-2 is prevalent in West Africa and produces a less
severe disease than does HIV-1 the most common form elsewhere.
The life cycle of the virus is described below in some detail since for a drug to be
effective it has to interfere with at least one stage of its life cycle. The HIV virus particle
is roughly spherical shaped and is about a thousandth of a millimetre across. Its outer
membrane consists of lipid molecules which posess many viral protein spikes projecting
outwards. Each spike is thought to consist of four molecules of glycoprotein gpl20 with
the same number of glycoprotein gp4l molecules embedded in the membrane itself.
These envelope proteins come into play when HIV binds and then enters target cells.
Gpl20 can bind tightly to CD4 proteins sited in the membranes of immune system cells
especially T lymphocytes also called T cells. This is the first stage of the infection which
is followed by fusion of the virus and T cell membrane, a process governed by the gp4l
envelope protein. The result is that the contents of the Virus core are thus freed to enter
the cell. The virus core is surrounded by matrix protein called p17 and is itself in the
shape of a hollow cone made of another protein p24 containing the genetic material of
the virus.
Being a retrovirus this genetic material is in the form of RNA (ribonucleic acid)
consisting of two RNA strands. These are in turn attached to molecules of an enzyme,
reverse transcriptase, which transcribes the viral RNA into DNA once virus has entered
the cell. Coexisting with RNA are an integrase, a protease, a ribonuclease and other
enzymes. Once in the cell the viral RNA is converted to DNA which then enters the cell
nucleus. The next step is integration of viral DNA into host chromosomes. This is
followed by cell proteins binding to DNA initiating transcription. Short RNA molecules
then leave the nucleus and make viral regulatory proteins followed by medium length
and long RNA which generate structural and enzymatic proteins. These assemble to form
new viruses (replication-viral budding) (1).
Prior to 1991 the only drug available to combat HIV/AIDS was Glaxo-
Wellcome’s AZT (zidovudine) a nucleoside analogue which works by binding to the
reverse transcriptase enzyme thereby inhibiting viral replication. Unfortunately, long
term use led to the virus developing resistance against the drug by mutation. New drugs
in the same class were subsequently developed including 3TC (lamivudine) (Glaxo-
IEUHUZI
Wellcome), ddc (zalcitabine) (Roche), dd] (didanosine)(Bristol-Myers Squibb), d4T
(stavudine) (Bristol-Myers Squibb) and recently abacavir (Glaxo-Wellcome).
1996 saw the introduction of a new class of drugs which acted at a different (and
later) stage in the HIV virus’ life cycle by blocking the action of the protease enzyme
during viral replication. Furthemiore, use of one of these with two of the class above
(reverse transcriptase) gave viral loads in the blood being reduced by up to 4 log units or
by a factor of ten thousand. Use of one drug alone reduces viral load by up to 2 log units
or by a factor of one hundred. An effective example of this so called triple therapy would
be use ofAZT and 3TC (reverse transcriptase inhibitors) and indinavir (Merck Sharp
and Dohme) or nelfinavir (Agouron) (protease inhibitors). Other protease inhibitors
include saquinavir (Roche), ritanovir (Abbott laboratories) and amprenavir (G1axo—
Wellcome). In general, effective therapies employ two reverse. transcriptase inhibitors
together with one protease inhibitor.
1996 also saw the introduction of another new class of drugs known as non-
nucleoside reverse transcriptase inhibitors, the first being nevirapine (Boehringer
Ingelheim) followed by delavirdine (Pharmacia Upjohn) in 1997 then efavirenz (Du
Pont) in 1998.
New effective therapies also capable of reducing viral loads by up to 4 log units
or by a factor of 10,000 employ a combination of nucleoside and non—nucleoside reverse
transcriptase inhibitors using a total of at least three drugs.
The cost of any triple therapy per patient per year is £10,000 -£15,000. (2)
The following table gives an overview of current AIDS drugs, their type or class,
effectiveness in reducing viral load, total amount of drug given to patient each day in
number of doses, side-effects, time for viral drug resistance to develop when used alone,
and approximate cost per patient per year. (2).
The first mentioned nucleoside reverse transcriptase enzyme inhibitor zidovudine
(AZT) when used by itself has subsequently been shown to provide no benefits in
treating HIV-infected individuals (3) although it is effective reducing transmission from
mother to baby (4).
However, it can be effective when used in conjunction with other AIDS drugs
such as 3TC, another nucleoside reverse transcriptase enzyme inhibitor (5).
.l()
lE01iu2t
Additionally, the HIV virus develops viral drug resistance against AZT rather
quickly (5-6 months) when used alone and even more rapidly (1 and a half months)
against 3TC when used alone (2). All nucleoside reverse transcriptase enzyme inhibitors
can cause serious side effects ranging from myopathy to peripheral neuropathy (nerve
damage). The most recent drug abacavir’s side effects can be life-threatening so
treatment with this drug is immediately stopped at the first signs of any adverse
reactions. Also ddc is a very toxic drug. Reduction in viral loads by drugs used on their
own are only moderate 50-90% and their cost is quite high (£1,200-£10,000 per patient
per year) (2).
The relatively recently developed non-nucleoside reverse transcriptase enzyme
inhibitor AIDS drugs can cause severe skin reaction in patients and the HIV virus can
develop viral drug resistance against them very quickly in 2 months in monotherapy (one
drug). In addition, cross viral drug resistance has been noted using this class of drugs. In
this case drug resistance against one drug in the class can cause drug resistance against
another drug of the same class (2). Again used by themselves they only reduced viral
load in patients by 50-90% and are relatively expensive (£1800-£2400 per person per
year) (2).
The new protease enzyme inhibitors have to be given to patients in relatively
large amounts (1250-2400mg per day) and can give serious side effects ranging from
kidney stones to hepatitis and after prolonged use patients exhibit raised levels of
cholesterol and triglycerides and can cause diabetes and abnormal distribution of body
fat. In addition they are expensive (£4000-£7000 per person per year) (2). They are also
generally poorly absorbed and have poor bioavailability which could well be related to
their low water solubility (6), (Protease Inhibitors in Patients with HIV disease by
M.Barry, S. Gibbons, D. Back and F. Mulcahy in Clinical Pharmacokinetics March 32
(3) 1997 p194) and can interact with other protease enzyme inhibitors and
nucleoside/non-nucleoside enzyme inhibitors in combination therapy, giving rise to a
very strict order of oral dosing which must be adhered to by the patient (7)
(Pharmacokinetics and Potential Interactions amongst Antiretroviral Agents used to treat
patients with HIV infection by M. Barry, F. Mulcahy, C. Merry, S. Gibbons and D. Back,
Clinical Pharmacokinetics, April 36(4) 1997 p289).
lElllll]2l
MARKETPLACE COMPARISON
DRUG TYPE ’ ‘REDUCTION TOTAL sum EFFECTS ' VIRAL DRUG ?osT/ 1
IN VIRAL AMOUNT RESISTANCE PATIENTI
LOAD DRUG/DAY (MTHS) YEAR
in (x) doses (PUNTS)
COMPANY
Zidovudine nucleoside 50-90% 600mg (2) . myelosupression, 5-6 £7,000-
(AZT) reverse myopathy, nausea, £10,000
transcriptase headache, anaemia Glaxo-
inhibitor Welleome
Lamivudine ‘ nueteosifie ' " so-90% 300mg(2) gastrointestinal 1'/2 £7,000
(3TC) reverse disturbances, hair Glaxo-
transcriptase loss, Welloome
enzyme myelosuppression,
inhibitor exacerbation of
peripheral
neuropathy
_Sravudine nucleoside S0-90% 40mg(2) peripheral greater than 6 £1,800
(d4T) reverse neuropathy Bristol
transeriptase Myers
enzyme Squibb
inhibitor
Didanosine nucleoside 50-903/I.‘ 300-400mg peripheral greater than 6 £2,000
(ddl) reverse (1) (at night) neuropathy, nausea Bristol
transcriptase vomiting, pancreatis Myers
enzyme l Squibb
V inhibitor
; Zalcitabine nucleoside 50-90% 0.75mg (1) ‘very severe : greater lhan 6 ' £1,200
(ddc) reverse (with meals} peripheral neuritis Roche
transcriptase
enzyme
inhibitor
Abacavir in % W0?/I 300mg(2) any reaction can be 0 £5,400
reverse life-threatening Glaxo-
transcriptase always stopped Wellcome
enzyme immediately
inhibitor
|E011t]2.I
DRUG TYPE REDUCTION TOTAL SII)E EFFECTS VIRAL DRUG COST!
IN VIRAL AMOUNT RESISTANCE PATIENTI
LOAD DRUG/DAY (MTHS) YEAR
in (X) doses (PUNTS)
COMPANY
Nevirapine hr? fi0% 200mg (2) ‘skin reaction . 2 £1,800
nucleoside Boehringcr
reverse Ingelheim
transeriplase
enzyme
inhibitor
HDelaviridine 0 non- 50-90‘? 600mg (3) skin reaction 2 — £1,800
nuelcoside many tablets Pharmacia-
reverse Upjohn
transcriptase (Agouron)
enzyme I
inhibitor
Efavirenz non- 50-90% 600mg (0 skin reaction 2 £2,400
nucleoside Dupont
reverse
transcriptase
enzyme
inhibitor
Indinavir _ - 500mg (3) hyperbilrubinaemia, 6 £5,000-
enzyme ‘ ’ nephrolthiasis, £7,000
inhibitor nausea, kidney Merck Sharp
stones, dizziness & Dohme
Ritonavir protease 99% 1800mg (2) . diarrhoea nausea, 6 K £5,000-
(not used by enzyme vomiting, hepatitis, £7,000
itself) inhibitor headache Abolt
Laboratories
K m? . _ 9?% 1800mg (2) loose stooT, natgas 6 £5,000-
enzyme headache £7,000
inhibitor Roche
u=_u1tu2t
DRUG TYPE REDUCTION TOTAL SIDE EFFECTS VIRAL COSTI
IN VIRAL AMOUNT DRUG PATIENTI
LOAD DRUG/DAY RESISTANC YEAR
in (x) doses E (MTHS) (PUNTS)
‘ COMPANY
Ncllinavir protease 7 99% ' l250mg (2) a di L1sea 6 £4,000-
(Viracept) enzyme l lot of tablets & vomiting £5,000
inhibitor l total 10 Agouron
l (Roche)
Amprenavir protease 99% i a lot of severe rash i - £7,000
(can be used enzyme tablets Glaxo-
with inhibitor Wellcome
Ritonavir)
0&1 protease enzyme inhibitors raise patients aiolesterol, triglyceride levels and can cause diabetes, kidney stones
and abnormal distribution of body fat after prolonged use.
The concentration at which an HIV-1 drug is effective is designated EC50 pm
which represents when the number of cells protected from HIV injection is half the total.
The antigen Agpl 20 assay - the virus related antigen - is related to the number of Virus
particles produced by measuring glycoprotein gp 120 in infected cell cultures. The
concentration of the drug which reduces cell growth by 50% is designated TC50uM.
Of course the lower the EC5o concentration the better but the real criterion of
effectiveness in in \/itjio testing on cell cultures is the Therapeutic index which is the
TC50/Ecso ratio. The therapeutic index is selected so as not to damage healthy cells.
Thus AZT has an EC50 ofg 0.016 1.1M with a TC5o>1000 pM. This results in a
therapeutic index of >1000/0.016 => 62,500. This figure serves as a benchmark against
which new potential drugs can be measured. Of course human beings and animals are
more than a collection of cells and in spite of the high Therapeutic Index, AZT is quite
toxic, giving rise to nerve damage and anaemia among other things (2). Nevertheless,
such tests on cell cultures indicate what is a potential anti-HIV drug.
Other factors relevant to the usefulness of an anti-HIV drug are physical
properties such as water-solubility for drug absorption by the patient and stability of the
IEDHBI1
compound after oral intake. Thus the potentially useful drug, the anionic polysaccharide,
dextran sulphate is poorly absorbed orally and degrades after oral intake before entry into
the plasma (8). Another important factor is the ease of synthesis of the drug and hence
drug cost which is relatively high for AZT and most other drugs produced to date which
are potentially useful in combating AIDS.
International Publication No. WO9403164 decribes compounds having
biological activity, particularly sulfonate based calixarenes, having anti-HIV activity.
The present application relates to compounds selected from the general group of
compounds disclosed in international application no. PCT/IE 95/00008 having especially
surprising activity. This application relates in particular to cyclic tetrameric pyr0gallol-
aldehyde derivatives and to calixarene derivatives which are useful in the treatment of
AIDS.
As mentioned previously, a high therapeutic index is required for an anti-HIV
drug to be effective. In particular international application no. PCT/[E95/00008 discloses
(AC-3 (Example 40 in PCTHE95/00008)) the potassium acetate of p-nitrocalixarene.
This compound has a relatively low therapeutic index of >375 (in vitro) as compared to
AZT which has a therapeutic index of >62,500. Such a comparison of values teaches
away from the use of this particular compound in phamiaceutical compositions for the
treatment of patients with HIV-1. Surprisingly, however, this compound appears to be
particularly effective as an anti—AlDS drug.
The present application also relates to the use of this compound in a
pharmaceutical composition for the treatment of HIV-1.
There is a need for an anti—HIV drug which brings about a reduction in viral load
but without causing the development of viral drug resistance and problems of toxicity. In
short, a drug is needed which when given orally gives rise to at least a M.I.C. (Minimum
inhibitory concentration) of drug in the blood against HIV but at a low enough
concentration so as not to give rise to adverse side effects in the patient.
IEUHUZI
Object of the Invention
It is an object of the present invention to provide novel and easily synthesised
compounds having biological activity, particularly anti-HIV activity, particularly against
HIV-1 .
It is a further object of the invention to provide compounds having a low EC5o or
MIC in patients blood (plasma) concentration which exhibit reduced and preferably little
or no side effects, and bring about a reduction in viral load but without causing the
development of viral drug resistance and pharmaceutical compositions thereof.
Summag of the Invention
The invention provides compounds of formula I
‘H41 /l»J'R|
/ L c Iu<__]., ._
1/ ‘\
Formula I
wherein R1 is CH2CO2K, CHZCOZH or CH-3CONH2 and R2 is
2.»: |g.< (I ll
where Hal is a halogen, preferably F or Br, and L is H or a halogen, preferably Br.
In a particular embodiment the invention provides compounds of formula I wherein
2n wherein R. is CH2CO2K or CH2CO2H and R2 is
IEUIIUII.
K /UL"ll3(‘()_.l|
Q for
and L is H or a halogen, preferably Br.
In another preferred embodiment the invention provides a compound wherein R1
lS CH2CO2K, R2 is
ll’ 1‘!
and L is Br.
Further preferably the invention provides a compound wherein R1 is CH2CO2H,
R2 iS
' l J | | I I | l
<_ ,: 1
and L is H.
Still further the invention provides a compound wherein R1 is CH2CO2K and R2
and L is Br.
The invention also provides a compound wherein R1 is CH2CONH2 and R2 is
n IEu11o§i
and L is Br.
The invention provides a method for the synthesis of a compound of Formula I
above as outlined in Examples 1, 2, 5 and 6.
The invention further provides a compound of formula II for use in the
preparation of a medicament for the treatment of viral infection, particularly HIV-1
infection.
.f"l.J:j]\CH“:
».\_ (H in-n.|\'
Formula II I
The compounds of formula I or H of the invention may be used in the preparation
of a medicament for the treatment of viral infection, particularly HIV-1 infection.
The invention further provides a pharmaceutical composition comprising a
pharmaceutically effective amount of a compound of formula I or II. The compounds of
the present invention may be used in combination with pharmaceutically acceptable
diluents or carriers to form pharmaceutical compositions for the treatment of viral
infections, particularly HIV -1 infection.
Preferably, the invention provides a pharmaceutical composition comprising
AZT and one or more of the compounds of formulae I and H.
IEu11u2I
The pharmaceutical composition according to the invention may comprise a
compound of the invention together with a pharmaceutically effective carrier or
excipient, and may be formulated as an inj ectable solution, a tablet, capsule, suppository
or as a cream, gel or ointment for topical application.
The medicament may be used in the prevention of HIV-1 by using it in
conjunction with a condom for example. The compounds of the invention have an
improved selectivity index, over commercial topical spermicides and disinfectants,
against HIV-1.
The invention provides a method of treatment of HIV infection comprising
administering to a patient a pharmaceutically effective amount of at least one compound
of formula I or H.
The invention also provides a method of treatment of HIV further comprising
administering to a patient a pharmaceutically effective amount of at least one compound
of formula I or II together with a pharmaceutically effective amount of AZT.
The invention will now be described in greater detail with reference to the
following Examples.
Detailed Descri tion of the Invention
Example 1 AC~1
(H II.( ()»I\'
K0)_CH2CO\ /l\\ /(’(‘ll»(" ’,vl‘§
.// ..'
(\_/ L I
/1 ('H_ I _ AC-1
/ llr \ "‘ I
F ,x
13 115011021
Step (i) Preparation of:
233g (l.88niole) of P-fluorobenzaldehyde was reacted with stirring with 236.5g (1.88
mole) pyrogallol in 1.11 absolute ethanol and 275 mls 37% aqueous hydrochloric acid
under reflux for 5 hours. After cooling the reaction mixture was filtered through
scintered glass and the grey—brown solid collected was washed once with 4:1 absolute
ethanolzwater then filtered again and left to dry overnight in the oven at 60°C to give
286.6g pyrogallol P-F-phenyl tetramer product in 65% yield.
Elemental analysis calculated for C52H35O12F4:
C = 67.24, H =3.91% Found C=66.84, H=4.00%
Step (ii) Preparation of:
H: ,-l i
ll [C 51
l43.3g (0.15 mole) pyrogallol P-F-phenyl tetramer was added to 21 glacial acetic acid to
which was added dropwise with stirring during 50 minutes lO6.0g (0.66moles) elemental
bromine under nitrogen at room temperature.
After addition the reaction mixture was stirred for 48 hours at room temperature
under nitrogen, following which it was poured into a large volume of water in a well-
ventilated area to precipitate the product.
The grey-brown solid was filtered through a grade 4 scintered glass funnel for
filtering fine small particle solids and then washed once with water reflltered and dried
in the dark for several days until perfectly dry at room temperature, occasionally
manually breaking up lumps. The solid turns pink in the light. It is important that the
product is perfectly dry at this point. Yield ofbromo-pyrogallol P-F-phenyl tetramer is
132.0g (69%).
Elemental analysis calculated for C52H32O12F4Br4: C=50.19%, I-l=2.59%
Found C=49.63% H=2.54%
Step (iii) Preparation of:
OCI goo-,.C.i cc: L
CH3CH2CO2C”“‘Og\ '/t)(}l'l;vCCJ;»('.l|;~(Llin
I K .
_/i\’\vl/ -..'tr:\——~
‘I ‘ ll: / \
/ / \
3’ if ll
.\ I
l60.0g (0.13mole) brorno-pyrogallol P-F-phenyl tetramer was reacted with 324g (2.3
mole) anhydrous potassium carbonate and 283g (1.69 mole) ethyl bromacetate (care
lachrymator) with stirring in 51 dry HPLC acetone refluxed for 3 days with drying tube
attached. After cooling to room temperature all volatiles were removed under reduced
pressure employing a rotary evaporator (11 of reaction mixture at a time) then residue
was treated with 230mls 37% aqueous hydrochloric acid mixed with 11 water. The
slightly sticky red-brown solid was then washed with water and transferred to a glass
dish in a 60°C oven. After a day or two at 60°C the product partially melts and water
separates out which can be poured off to accelerate drying. Afier several more days the
product hardens as it dries. This normally takes several more days. It is important that
the solid is dry. When the solid is dry it is pulverised with a mortar and pestle to give
247g (80% yield) of the dodecaethylacetate of p-bromopyrogallol P-F-phenyl tetramer.
\Eu11021
|El]l1[]2l
Elemental Analysis Calculated for C;ooH[o4036F4Br4: C=52.73%, H=4.60%, Found
C=S2.36%, H=4.23%.
Step (iv) Preparation of:
1M'll.( ().}\'
K02CCH;:‘?‘ ~'Ir«‘<~ :4
247g of the dodecaethylacetate of p-bromopyrogallol P-F-phenyl tetramer is stirred for 2
hours under reflux with drying tube attached with 200g KOH in 21 absolute ethanol then
filtered hot through scintered glass and the red-brown solid immediately placed in a 60°C
oven for 4 hours to give 260g product 100% yield of dodecapotassium acetate of p-
bromopyrogallol P-F-phenyl tetramer as a red brown solid. AC-1
This product is somewhat hygroscopic and filtering hot and immediate transfer to 60°C
oven renders a product much less prone to becoming sticky in the air than filtering at
room temperature and leaving in the open air to dry at room temperature.
Elemental Analysis Calculated for C75H44O36F4Br4K12:
C=38.06% H =1 .85%. Found C=38.07, H=2.30%
Example 2 AC-2
0cH;;Co;,H
HO;;CCH-_;O ,OCH;,COgH
- .:\(:.>_
CH3‘ »— \
OCH2COyH 3
'\ /’
L 1,,’/'
lE0ll|]2l
Step (i): Preparation of :
/.' __ _4£')(.li-(,I\'_3»§'-i
6.31 g (0.05mo1e) pyrogallol and 9.01 g (0.05mole) 2-fonnylphenoxy acetic acid were
reacted together with stirring in 40mls absolute ethanol and 1011115 37% aqueous
hydrochloric acid under reflux for 4 hours. After cooling the reaction mixture was
filtered through scintered glass and the brown-grey solid was then washed with absolute
ethanokwater 4:1 then filtered and dried overnight at 60°C in an oven to give 10.4g (72%
yield) of pyrogallol 2-phenoxyacetic acid tetramer.
Elemental analysis calculated for C50H43O242
C=62.50, H=4.20%, Found C=62.01, H=4.l0%
Step (ii): Preparation of:
'7”,-HQCOQCHQCH3
H3CCH2CO2C-‘ ‘--~\' “_ I 4 " '-H2CO2CH2CH3
lp lie all 1
_ ‘ {Alli _|
\_,OCH2CO2H
M0 ii 9]
.\‘_:/ ‘
To 10.4g pyrogallol 2-phenoxyacetic acid tetramer (0.009 mole) was added 30.3g (0.220
mole) anhydrous potassium carbonate and l9.9g (0.120 mole) ethyl bromoacetate (care
lachrymator) in 390mls dry HPLC acetone and the entire was refluxed for 3 days with
fl’)
|E0lll]2l
diying tube attached. After this time all volatiles were removed and to the residue was
added 22mls 37% aqueous hydrochloric acid mixed with 100mls water.
After breaking up the solid and stirring well the reaction mixture was filtered through
scintered glass and then washed with water filtered and dried i11 60°C oven for 5 days to
give dodecaethyl acetate of pyrogallol 2-phenoxyacetic acid tetramer 14.3g (73% yield).
Elemental Analysis calculated for C1031-1120043 C=59.33, H=5.53%, Found C=59.l0,
H=5.2l%
Step (iii): Preparation of:
<‘>(':I l,.(;.(>_,|—l
llO;«;3l 131:0. ~ .<')(:l i;.t:c.1_~.ii
?i©ii
§[ C _
/OCl'l2CO2l"l
143;; (0.06 mole) dodecaethyl acetate of pyrogallol 2-phenoxyacetic acid tetramer was
added to 14.3 g KOH in 140ml absolute ethanol and the entire was refluxed with drying
tube attached for 31/2 hours. After filtering hot through scintered glass the solid* was
immediately added to 501111 glacial acetic acid in l75mls absolute ethanol with stirring
and filtered through scintered glass then immediately placed in 60°C oven for several
hours to give l2.0g (99% yield) dodeca-acetic acid of pyrogallolphenoxyacetic acid
tetramer AC-2 which is very hygroscopic (picks up moisture from air) and should be
stored in sealed container as a purple brown solid quickly becoming sticky in the air. The
product contains some potassium acetate from the neutralisation of the hexadeca
potassium acetate pyrogallol tetramer with acetic acid
Elemental analysis calculated for C34H7204g.l6CH3CO2KI
C=40.74, H=3.54% Found C=40.30, H=3.8l%
|Eu11l1Zl
* A small sample was added to 20% aqueous HCI which was then placed in a fridge.
After one week colourless crystals of pure product were obtained.
Elemental Analysis calculated for Cs4H72043.6H20Z
C=5l.53, H=4.32% Found C=51.07, H=4.32%
Example 3 - AC-3 Route A
0‘. ii l_ (Tl ‘' »l\'
Step (i): Preparation of:
([92:05/]\" ":‘l"' ‘
on L’
A slurry of 13.3g (0.020 mole) p-t-butylcalixarene, 9.02g (0.096 mole) phenol* and
14g (0.015 mole) anhydrous AlC13 was stirred in 12Smls toluene at room temperature for
1 hour under nitrogen with drying tube attached. The mixture was then poured into
250mls 0.2N HCl, the organic phase was separated and toluene was removed under
reduced pressure on a rotary evaporator. Upon the addition of methanol a precipitate
formed which was removed by filtration to give 7.54g 89% yield calixarene as an off-
white solid which was dried well in an oven at 60°C for several hours. It is important that
the product is dry (dry overnight 60°C).
*May be left out but the reaction time is then 1 day at room temperature. Ref:
C.D.Gutsche and L.Lin Tetrahedron 42 6 1986 pl633
Elemental analysis calculated for C23H24O4Z C=79.22, H=S.70% Found C=79.10,
=5.60%
Step (ii): Preparation of:
19 |E011l]21
I (1 H; i »
C )1 I ‘ 4
.0g (0.012mole) ca1ix—4-arene was stirred in 5011315 concentrated sulphuric acid at 95°-
l00°C for 10 hours. After cooling the reaction mixture was diluted with 90mls cold
water (external cooling required - care) and then entire was cooled to 0°C and then
treated with 5.8g 61% HNO3 for 10 hours at 0°C with stirring. The entire was then
diluted with water Q 5001111 and a fine yellow solid product precipitated which was
filtered off then washed once with water then dried in 60°C for several hours.
It is important that the product is dry. Yield =7.1g (99% yield) as a pale yellow green
solid p-nitrocalixarene. (Dry in oven at 60°C overnight).
Procedure adapted from S.Shinkai, T.Tsubaki, T. Sone and O.Manabe, Tetrahedron
Letters 26(28) p3343 1935
Elemental analysis calculated for C23H2oN4O12
C=55.63, H=3.34% Found C=55.l3, H=3.66%
Step (iii) Preparation of:
NO,_-
(H2 e‘ 7“
OCH2C|iOCH2CH3
11.2g (0.019 mole) p-nitrocalixarene was added to 15.5 g (0.11 mole) anhydrous
granular potassium carbonate, 13.0g (0.078 mole) ethyl bromoacetate (care lachrymator)
and 200mls dry HPLC acetone and the entire was refluxed with drying-tube attached for
(E u l 1 Q 4 1
three days. After cooling all volatiles were removed under reduced pressure on a rotary
evaporator and to residue was added 110mls 10% aqueous HCl (hydrochloric acid). The
solid was well broken up and stirred well then filtered off and the solid was washed with
water then dried for several hours in an oven at 60°C.
It is important to dry the product thoroughly again. Yield l5.7g (87%). The tetraethyl
acetate of p-nitroealixarene (dry 60°C oven overnight).
Elemental analysis calculated for C44H44N4O20.5H2O. C=5 l .26, I-I=4.50% Found
C=51.25, H=3.99%
Step (iv) Preparation of:
r[q<_)«_.
c: H; \
(.\ OCH;-COgK 4 )
-cs» -
l7.2g (0.018 mole) tetraethylacetate of p-nitrocalix—arene was refluxed with 17g KOH
in 1 1 lg absolute ethanol for two hours with drying tube attached. After this time the
reaction mixture was filtered through scintered glass while still hot to give 17.7g (99%)
of potassium acetate of p-nitrocalixarene as a red-brown solid AC-3 which was dried
in an oven at 60°C for a few hours.
Elemental analysis calculated for C3(,I-lg4N4O20K4: C=43.72, I-I=2.45% Found C=43.37,
H=2.90%
Example 4 AC-3 - Route B
To 2.99g (0.003 mole) t-butylcalix-4—arer1e tetraethyl acetate in 30 mls dichloromethane
and 30mls glacial acetic acid cooled to and kept at 0°C was added with stirring 10ml
(0.240 mole) 100% nitric acid HNO3. The reaction mixture was stirred then for 40
minutes at 0°C and subsequently allowed to warm to room temperature and stirred until
the black-purple colour had discharged. It was then poured into 200mls water. The water
layer was extracted with 2 x 50mls dichloromethane. The combined organic layers were
QEUIIBZI
washed with water 2 x 30mls then dried with dried magnesium sulphate then volatiles
removed to give product which was then recrystallised from dichloromethane/petroleum
ether 40°C-60°C to give l.06g (37% yield*) p-nitrocalixarene tetraethyl acetate which
was converted into AC-3 using the procedure as outlined in method A, step (iv).
*Ref: W. Verboom, A. Durie, R.J.M. Egberink, Z. Asfari and D.N. Reinhoudt, J. Org.
Chem. 57 1992 p13l3.
22 |Eu1tu21
Example 5
240g (1.30 mole) of P~brornobenzaldehyde (1) was reacted with stirring with 163.5g
(1.30 mole) pyrogallol (2) in 1.5 litres absolute ethanol and 2l5mls 37% aqueous
hydrochloric acid under reflux for 4 hours. After cooling the reaction mixture was
filtered through scintered glass and the brown solid collected and washed with 4:]
ethanoltwater before drying at 70°C to give 266g pyrogallol P-Br-phenyl hexanrer
product (3) in 70% yield.
l00g (0.06 mole) of P-Br-phenyl hexamer (3) was added to one litre glacial acetic acid to
which was added drop wise with stirring during 30 minutes 642;; (0.4 moles) elemental
bromine under nitrogen at room temperature. After addition the reaction mixture as
stirred for 24 hrs at room temperature under nitrogen, following which it was poured into
a large volume of water in a well-ventilated area to precipitate the product. The brown
solid was filtered through a grade 4 scintered glass funnel for filtering fine small particle
solids and then washed once with water refiltered and dried in the dark at room
temperature. Yield of bromo-pyrogallol P-Br-phenyl hexarner (4) is 89g (60%).
80g (0.04 mole) Bromo—pyrogallol P—Br~phenyl hexamcr (4) was reacted with l 13 g (0.8
mole) anhydrous potassium carbonate and l30.2g (0.78 mole) ethylbromoacetate (5)
with stirring in 2 litres of HPLC grade acetone and refluxed for 24 hours. After cooling
to room temperature all Volatiles were removed under reduced pressure employing a
rotary evaporator and the residue treated with l50mls 37% aqueous hydrochloric acid
mixed with 80011115 of water. The resultant brown product was dried at 60°C to give
128.5 g (85% yield) of the ethylacetate of p-bromopyrogallol P-Br-phenyl hexamer (6).
128 g (0.034 mole) of the ethylacetate of p-bromopyrogallol P-Br phenyl hexamer (6) is
stirred for two hours under reflux with 48g KOH in one litre absolute ethanol and
filtered hot through scintered glass. The resulting brown solid of the potassium acetate
salt of p-bromopyrogallol P-Br-phenyl tetramer (7) (95% yield - 144g) is dried at 60°C.
H I Br; Br
H OH
':I «Q
6 BrcH2co2cH2c19
(5L4°”
(4) 9°
3‘ ‘ll2C02CH2Cll3
Br 06°
cnaicnzcogcl-lg CH2C02CHzC!»I3
<2°(<
24 150111121
Example 6
250g (1.35 mole) of P-bromobenzaldehyde (1) was reacted with stirring with 170.1 g
(1.35 mole) pyrogallol (2) in 1.7 litres absolute ethanol and 225mIs 37% aqueous
hydrochloric acid under reflux for 3 hours. After cooling the reaction mixture was
filtered through scintered glass and the brown solid collected and washed with 4:1
ethanolzwater before drying at 70°C to give 297g pyrogallol P-Br-phenyl tetramer
product (3) in 75% yield.
100g (0.085 mole) of P-Br-phenyl tetramer (3) was added to 800m1s of glacial acetic
acid to which was added drop wise with stirring during 30 minutes 29.5 g (0.0187 moles)
elemental bromine under nitrogen at room temperature. After addition the reaction
mixture was stirred for 36 hrs at room temperature under nitrogen, following which it
was poured into a large volume of water to precipitate the product. The brown solid was
filtered through a grade 4 scintered glass funnel for filtering fine small particle solids and
then washed once with water, refiltered and dried in the dark for several days until dry.
Yield of bromo-pyrogallol P-Br-phenyl tetramer (4) to 89g (70%).
80g (0.054 mole) Bromo-pyrogallol P-Br-phenyl tetramer (4) was reacted with 1 13 g
(0.713 mole) anhydrous potassium carbonate and 96.6g (0.700 mole) 2-bromoacetamide
(5) with stirring in 1.5 litres of HPLC grade acetone and refluxed for 12 hours. After
cooling to room temperature all volatiles were removed under reduced pressure
employing a rotary evaporator and the residue treated with 100mls 37% aqueous
hydrochloric acid mixed with 400mls of water. The resultant brown product was dried
at 60°C to give 93.8g (80% yield) of the dodecaacetamide of p-bromopyrogallol P-Br-
phenyl tetramer (6).
u-3011021
Egatnnlg Q
.110 H “
no on H H
9 TD
Bf . .
‘ 1) CH
( H ‘Bk
H( H
<3‘ ')Cl-l3CONH2
Br 4
‘all
26 |EUl1D2l
Clinical Results
Anti-HIV Activig;
Detennination of ECE and TC5_Q
Antiviral Assays
The anti-HIV and anti-SIV (Simian immunodeficiency Virus) activities and
toxicities of compounds were assessed in C8166 cells infected with HIV-1 “[3, HIV -
LZROD, SIVMAC. The cells are cultured in RPM1 1640 with 10% calf serum.
Aliquots of 4 x 104 cells per rnicrotiter plate well were mixed with 5 fold
dilutions of compounds prior to addition of 10 CClD50 units of virus and incubated for
-6 days. Formation of syncytia was examined from 2 days post—infection. Gp120
antigen produced at 5-6 days was measured by ELISA, using the lectin GNA (from
Galanthus nivalis) to capture the glycoprotein and human anti-HIV serum for detection
(9). Cell viability of infected, and uninfected control cells was measured by the MTT-
Foimazan method (10).
Briefly, 10 ul of a solution of 3-(4,5-dimethylthiazol—2—YL)-2,5 dipheny1-
tetrazolium bromide (MTT, 7.5 mg/ml in PBS) was added to each well containing 100 pl
of infected or uninfected cells. After incubating at 37°C for 1 hour, the blue formazan
crystals produced are solubilized in 150 ttl of 10% V/V triton X-100 in acidified
isopropanol (2m1 concentrated HC1 per 500ml solvent) and absorbance read at 540nm.
pp 120 Antigen Assay
A mierotiter antigen capture ELISA was developed using lectin (GNA) from
Galanthus nivalis (Vector Laboratories, Peterborough, UK.) and human antibodies (10).
The plates were coated with lectin (0.5 ug), and after blocking with 10% calf serum,
dilutions of virus supernatant in 0.25% detergent solution (Empigen, Albright and
Wilson Ltd., Whitehaven, U.K.) were added to the wells and incubated at 4°C for 12-16
hours. Bound antigen was captured using human anti-HIV antibodies, and finally
detected with anti~human lg antibodies conjugated to horseradish peroxidase.
' AC-3 1
EC5o represents the concentration which reduces the Ag gp120 by 50% in infected cell
cultures.
TC50 represents the concentration of drug which reduces cell growth by 50%.
Table 1
Compound I ECSQ gm ' E V TC59zE;C5g gm
' AZT 0.016 >1000 T $62,500
' AC-1 0.1 ' 400 ‘4,000
' AC-2 1-2 100 50-100
>100 T T
>100
A comparison of the Therapeutic Index of the compounds of the invention with
that of AZT would suggest that these compounds especially AC-2 and AC-3 are not
worth testing in vii and actually teaches away from the invention. In general
researchers like to see a Therapeutic Index of thousands before progressing a drug to
clinical trial. However, the present inventor has surprisingly found that these compounds
are significantly more effective in vivo than AZT.
In vitro studies anti-HIV—l activity
An additive effect with AZT was demonstrated for AC-1, AC-2 and AC-3. AZT inhibits
reverse transcriptase enzyme. The drugs of the present invention inhibit fusion and
integrase enzyme. Also there was an additive effect for the three combinations of two of
the drugs of the present invention in vitro. Table 2 illustrates the additive effect.
All three drugs are believed to act against HIV-1 in the “fusion” stage of its life cycle.
28 15011021
TLbie;
Combination of Compounds ggmm
C AC-1 + AC-2 0.05 + 0.7
AC—1 + AC-3 0.04 + 0.5 _
AC-'2 + AC—3 0.7 + 0.5
— AC-1 + AZT 0.03 + 0.005
AC-2 + AZT 0.5 + 0.007
AC—3 + AZT 0.4 + 0.006 ‘
fltble. 3
lntegrase activity (Ref N.Neamati, S.Sunder and Y. Pommier, Drug Discovery Today 2
(1 1) 1997 p487)
Integrase Activity EC5g(pm) /3'Pr0cessin;
AC-3 C62
AC-1 14.3
AC-2 15.9
Each drug was administered orally to patients at a level of 500mg per day (three
doses of 167mg each 8 hours). It was found that each drug reduced viral loads in the
patients blood by an average of 2 log units i.e. 100 times. The viral load was measured
using a Roche Amplicor. A Roche Amplicor is a commercial apparatus for measuring
viral load in patients blood measured in copies per ml of blood which means the number
of free HIV-1 viral particles per millilitre of patient’s blood. This quantitative method is
based on PCR (Polymerase Chain Reaction) and is FDA-approved.
All patients also registered a significant increase in CD4 expressing cells which
is an indication of improved immune status. A Becton Dickinson Flowcytometer was
used to measure the level of CD4 cells. This apparatus is commercially available and is
used to measure levels of cells in the blood including CD4 T cellls. A blood sample of
known volume is taken from the patient and the white blood cells are separated off.
These are then suspended in a liquid to which are added flourescent-labelled monoclonal
antibodies specific for the type of blood cell whose level is needed to be measured e.g.
\\=_tH.lll2.l
CD4 T cells, to which they become attached by binding to the specific protein present in
the cell such as CD4. The result is a suspension of flourescent-labelled-CD4 cells in this
case which are passed through an exceedingly fine nozzle in the Becton Dickinson
F lowcytometer which is electrically charged and which allows only one flourescent —
labelled cell to pass at a time which are then directed to a flourescent light detector-
analyser attached to a computer. The total light change detected arising from the total
number of cells present reveals the number of flourescent-labelled CD4 T cells and
hence. the number of CD4 T cells present per ml of blood.
The patients treated using the drugs of the present invention were all with an
advanced stage of the disease. In particular, one female patient had, prior to treatment,
500,000 viral copies (free viral particles of HIV-1) per ml of blood. After treatment with
AC—l her viral loads were reduced to 1% of the original) and were maintained for at least
a year and were found to be fairly constant during this time period. A man with 750,000
viral copies per ml of blood was found 3 months later after treatment with AC-1 to have
50,000 viral copies with great improvement in clinical condition. Patients being treated
with the drugs of the present invention did not experience any side effects. Furthermore,
their clinical condition dramatically improved in all cases.
In addition, there was a significantly reduced incidence of opportunistic
infections and improvement in physical fitness (Karnofsky Score) in all patients. The
“Kan1ofsky score” is a measure of physical fitness on a scale of 0 to 100. A score of 0
refers to a person being dead and a score of 100 is a ‘normally’ fully fit individual with
all his faculties in order able to work full—ti111e and carry out activities associated with a
‘normal’ lifestyle. A disabled individual unable to walk and confined to a wheel-chair
with normal sight and hearing would merit a score of about 70. An individual on a
“ventilator” i.e. with his breathing needing assistance and in “intensive care” would be
given a score of about 30.
The drugs according to the present invention inhibit fusion and integrase enzyme.
Table 4 In vitro efficiency tests
EC5o represents_t-he aentration which reduces the viral_antigen p24 50% in infected cell
cultures.
TC” represents the concentration of drug which reduces cell growth by 50%.
Compound Conc Syncytia Antigen p24 Estimated Cell Growth EC5o TC” SI
Um (+/-) % Control % of Control TC/EC
Infected Uninfected
1 151C 400 73 69 0.25 500 2000
AC-l 80 105 100
(~60% 16 92
purity) 3.2 - 92
0.64 -.I'+ 16.8 72
0.128 ./+ 63 30
_0.0256 + 28 A .
04-002 400 T50 41 42 0.015 350 I 23333
AC-I 80 93 94
(~80% 16 99
purity) 3.2 104
0.64 101
0.128 - 14 100
0.0256 -/+ 30 46.5 __H' i an i _
04-003 400 T50 44 45 0.012 350 29166
AC-1 80 - 100 100
(-85% 16 96
purity) 3.2 100
0.64 100
0.128 - 0 100
0.0256 -/+ 25 55
04-004 400 T40 41 41 0.32 350 1100
Product 80 - 100 100
Inter 100
mediate 1 3.2 — 94
0.64 27 70
0.128 H'- 99 34
0.0256 ,3; 29
AC 200 T 16 16 8 30 3.75
Product 40 T25 29 3 1
I n ter— 8 «,/+ 5 4 5 8 98
mediate 2 1.6 ~ 99 27 100
0.32 27
i _ 0.064 ‘r 24 _ _
AZT 2 - 100 100 0.016 >1000 >62,500
Afi 0.016 ‘ +/- . 51 49 _
Control 0 + J 100 24 100 _ _
|E0ll02l
Column 3 = Syncytia is examined microscopically where infected cells fiise and produce
distinct giant cells.
Column 4 = Drug treated infected cell supernatant is removed and virus related antigen p24 is
measured by ELISA. Untreated infected control is taken as 100%.
Column 5 = Cell growth (infected and uninfected) is measured by MTT-formazan method.
Higher number of cells in infected cultures indicates protective effect of compounds and
uninfected cell controls show drug toxicity.
Compounds 04-002, 04-003, 04-004 and 1151C were dissolved in water at 20 mM diluted 1/10
in growth medium for testing.
Compound AC- was dissolved in DMSO at 50 mM and diluted 1/100 in medium for testing,
DMSO is not toxic to cells at 1% concentration.
Compounds 04-002 and 003 are at least 10 times better than 1151C and 04-004.
32 lE@lltlZl
The drugs demonstrated an improved performance over that known for AZT.
AZT is known to reduce viral loads in patients by 50-90%, that is up to 1 log unit (ten
times). However continued oral administration of AZT leads to quickly leads to the
development of viral drug resistance.
The drugs of the present invention bring about a reduction in viral load similar to
that exhibited by daily oral monotherapy with Merck Sharp and D0hme’s protease
inhibitor ‘indinavir’ (three doses of 800mg each 8hot1rs i.e. 2400 mg per day - much
higher dose needed!), or Hoffmann-La Roche’s protease inhibitor ‘saquinavir’ (three
doses of 600 mg each 8 hours i.e. 1800 mg per day) or ‘ritonavir’ (three doses of 600 mg
each 8 hours i.e. 1 800 mg per day). However, the drugs of the present invention do not
exhibit the 6 month development of viral drug resistance which accompanies the use of
these protease inhibitors when used alone i.e. in monotherapy. Therefore, protease
inhibitors have to be used in combination with others to prevent development of viral
drug resistance.
During a year’s treatment with the drugs of the present invention viral loads were
reduced by up to 99%. Unlike the protease inhibitors no side effects were observed.
Additionally, no viral drug resistance was observed during daily oral
administration of each drug over a period of a year ( 5 00mg a day).
This could well be due to the fact that the compounds of the present invention
appear to act at two different stages in the life cycle of the HIV-1 virus. The drugs of the
present invention appear to act on the early fusion stage and later integrase stage of the
life cycle.
An important advantage of the compounds of the present invention is their
relatively low cost and ease of synthesis. They are especially suitable for use in the
developing world.
The drugs of the present invention offer advantages over those of the prior art in
that they are lower in cost and a lower dose of drug is required. The high water solubility
IEIJHUZI
may give advantages over protease enzyme inhibitors in that they have superior
absorption by the body and a higher degree of bioavailability.
In addition use of only one drug avoids the complications of a strict regime of the
order of taking a combination of drugs which must be strictly adhered to by the patient.
All patients treated with AC-1, AC-2 and AC-3 experienced increased CD4 T cell levels
after treatment.
MARKETPLACE COMPARISON
DRUG TYPE REDUCTION TOTAL SIDE VIRAL DRUG COST!
(NEW) IN VIRAL AMOUNT EFFECTS RESISTANCE PATIENT/YEAR
LOAD DRUG/DAY (MTHS) (PUNTS)
in ()4) doses COMPANY
LAC-1 (fusion, 99% 500mg (3) none‘ ' none Aidsare
integrase observed observed Pharma Ltd.
enzyme during one during one
inhibitor year year
AC-2 fiision, 99%/ I 3I)0mg(3) I ?ne> ‘none Aids Care
integrase observed observed l Pharma Ltd.
enzyme during one during one
inhibitor year year
AC-3 fusion, 1 99% 500mg(3) none i none Aids Care
integrase observed observed Pharma Ltd.
enzyme during one during one
inhibitor year year
Viricidal Anti-HIV Activitv
AC-1, AC-2 and AC-3 were tested as vaginal viricides against HIV-1. An effective
vaginal viricide would need to have high anti-HIV-1 activity, low toxicity, stability at
relatively high ambient temperatures and at pH 4-7, absence of odour and taste and
cheapness (Private Communication from H.Pask, AIDS Secretariat Medical Research
Council, London, England). The drugs of the present invention were tested following the
method of T.J. O’Connor, D. Kinchington, H.O. Kangro and D.J. Jeffries, ‘The Activity
of Candidate Viricidal Agents low pH and Genital Secretions against HIV-1 ‘in vitro’ in
International Journal of STD and AIDS Q 1995 p267.
Compound IC5gum CC5gpm CC50pm/ ICs0IJIn
Selectivity Index
AC-1 ’1.1 I ">50 ‘>43.4
AC-2 ' 5.5 >50 >9
AC-3 5.5 >50 >9
nonoxynol-9 8 1 1 1.6
octoxynol 0.1 lug/ml 0.31ug/ml 2.8
benzalkonium 0.12ug/ml 0.60 pg/ml 5.0
chloride
chlorhehidine 0.25 1.3 5.2
lC5() is the concentration of compound giving 50% inhibition of HIV-1 p24 antigen
CC50 is the concentration of compound giving 50% of cell growth (cytotoxicity)
nonoxynol-9 is a commercial spermicide of Cilag Ltd.
octoxynol is a commercial spermicide from ICI surfactants
benzalkonium chloride is a commercial spermicide from Fluka
chlorhexidine is a commercial disinfectant from Sigma
The selectivity index of A01, AC-2, AC-3 is >43.4, >9, >9 which is superior to
the three commercial spermicides: nonoxynol-9, octoxynol and benzalkonium chloride
1.6, 2.8 and 5.0 and that of the disinfectant chlorhexidine 5.2.
The words “comprises/comprising” and the words “having/ including” when
used herein with reference to the present invention are used to specify the presence of
stated features, integers, steps or components but does not preclude the presence or
addition of one or more other features, integers, steps, components or groups thereof.
References
1. AIDS and the Immune System by W.C. Greene Scientific American 1993 p67.
2. Drug Cocktails Fight HIV by L.Gopinath, Chemistry in Britain June 1997 p38
and personal communications from Dr. Sam McConkey, Senior lecturer, Department of
Medicine, Oxford University, United Kingdom and Dr. Peter Mugyenyi, Director Joint
Clinical Research Centre, Kampala Uganda.
3. AZT Benefit in Doubt, Chemistry in Britain, May 1993, p62.
4. Defeating AIDS:What will it take'?, Scientific American, July 1998, p62.
. Potential Mechanism for Sustained Antiretro-viral Efficacy of AZT-3TC
Combination Therapy by B.A. Larder, S.D. Kemp and P.R. Harrigan. Science vol. 269, 4
August 1995, p696.
6. Protease Inhibitors in Patients with HIV Disease by M. Barry, S. Gibbons, D.
Back and F. Mulcahy in Clinical Pharmacokinetics, March 32(3) 1997 p194.
7. Pharmacokinetics and Potential Interactions amongst antiretroviral agents used to
treat patients with HIV infection by M. Barry, F. Mulcahy, C. Merry, S. Gibbons and D.
Back, Clinical Pharmacokinetics, April 36(4) 1999 p289.
8. Molecular Targets for AIDS therapy by H. Mitsuya, R. Yarchoan and S. Broder,
Science 28 September 1990 pl 533.
9. N. Mahmood, AJ. Hay (1992) An ELISA utilizing immobilised snowdrop lectin
GNA for the detection of envelope glycoproteins of HIV and SIV. J. Immunol Methods
151 :9-13.
. R. Pauwels, J. Balazarini, M. Baba, R. Snoeck, D. Schols,p.Herdewijn, J.
Desmyter and E. De Clerq, (1988) Rapid and automated tetrazolium based colorimetric
assay for the detection of anti-HIV compounds. J. Virol Methods 20:309-321.
“ 15011021
Claims (5)
1. A compound of formula I Formula I wherein R1 is CH;COgK, CH2CO2H or CHZCONI-lg and R2 is (‘)(_‘l|-_»(‘()-_-ll where Hal is a halogen, preferably F or Br, and L is H or a halogen, preferably Br.
2. A compound of Formula I as claimed in claim 1 selected from those in which 21) R1 lS CH2CO2K OI‘ CHQCOQH, R2 ls ,l l ‘W u'Il_.( ‘(MI and L is H or a halogen, preferably Br; b) R] is CH2CO2K, R2 i5 and L is Br; C) R1 is CH2CO2H, R2 iS andLisH; d) R; is CHZCOZK and R2 is and L is Br B) R1 is CHZCONHQ and R2 is and L is Br. IEUIYEIZ}
3. Use of a compound of formula I or II nu. Nu, Formula I Formula II wherein R; is CH2CO2K, CHQCOQH or CH2CONHg and R2 is where Hal is a halogen, preferably F or Br, and L is H or a halogen, preferably Br, in the preparation of a medicament for the treatment of viral infection, particularly HIV-1 infection.
4. A pharmaceutical composition comprising a pharmaceutically effective amount of one or more of a compound of formula I or formula II as defined herein or AZT, together with a pharmaceutically acceptable carrier or diluent.
5. A method for the synthesis of a compound according to claim 2(b) or 2(d) comprising the steps of (i) condensing pyrogallol with aldehyde in equimolar quantities in refluxing 37% aqueous HCI/ethanol to form a halogenated oligomer, washing the precipitated oligomer with 4:1 ethanolzwater and drying; (ii) adding bromine dropwise to said oligomer in glacial acetic acid under nitrogen at room temperature (iii) following addition of bromine, stirring reaction mixture at room temperature under nitrogen; pouring reaction mixture into water to precipitate the product; filtering the [E u 1 1 0 2 1 product to yield bromo-pyrogallol P-Hal-phenyl oligomer washing, refiltering and drying, preferably in the dark; (iv) reacting said bromo-pyrogallol P-Hal-phenyl oligomer with anhydrous potassium carbonate and ethyl bromacetate in refluxing dry acetone, allowing to cool to room temperature removing all volatiles under reduced pressure; (v) treating residue with 37% aqueous HCI mixed with water, drying, pulverising to form dodecaethylacetate of p-bromopyrogallol P-Hal- phenyl oligomer (vi) stirring dodecaethylacetate of p-bromopyrogallol P-Hal- phenyl oligomer with KOH in refluxing ethanol to yield dodecapotassium acetate of p-bromopyrogallol P-Ha1- phenyl oligomer, preferably the said oligomer comprising 4 or 6 monomer units, and Hal preferably being F or Br. TOMKINS & C0
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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IEIRELAND01/12/20002000/0983 |
Publications (2)
Publication Number | Publication Date |
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IE20011021U1 true IE20011021U1 (en) | 2002-12-11 |
IES83658Y1 IES83658Y1 (en) | 2004-11-03 |
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